Use of the oral antiapoptotic agent Flupirtine as a treatment strategy in human INCL, LINCL, JNCL and CLN6-deficient vLINCL

APOPTOTIC DEFECTS IN NCLAnti-apoptotic effects of intact CLN3 is firmly established in humans: 1. By EM and TUNEL of human JNCL brain (Lane et al,. 96).2. By JC-1, propidium iodide, TUNEL and LDH release of CLN3-deficient human neurons (Dhar et al., 2002).3. Resistance of stable CLN3-overexpressing NT2 cells to apoptotic death resulting from serum starvation and chemotherapeutic agents. Inhibition of etoposideinduced PARP cleavage, and therefore caspase-3 activation in these cells (Puranam et al,. 99).4. Increased apoptotic death of CLN3-deficient, naturally occurring patient derived fibroblasts and lymphoblasts resulting in net slower growth (Persaud-Sawin et al, 02). 5. Upregulation of CLN3 protein and RNA in a number of human cancer cell lines and solid colon cancer, as well as mouse breast cancer cells. Inhibition of cancer cell growth and initiation of apoptosis after blunting of CLN3 protein expression using antisense strategies (Rylova et al, 02).6. Indirect evidence: developmental regulation of CLN3, upregulation of Bcl-2 in surviving neurons by immunocytochemistry, Western blot, RT-PCR and Affymetrix Genechip analysis. Upregulation of many other neuroprotective genes by Affy Genechip analysis of human, CLN3-deficient fibroblasts (Pane et al, 99; Puranam et al, 97, unpublished).7. Increased ceramide in JNCL brain and JNCL fibroblasts and downregulation of ceramide levels in stable CLN3 overexpressing cells (Puranam et al, 97, Rylova et al, 02). 8. Recognition of anti-apoptotic motifs within the CLN3 protein (Persaud-Sawin et al, 02).

APOPTOTIC DEFECTS IN NCLEstablished in LINCL: 1. By EM and TUNEL of human LINCL brain (Lane et al, 96).2. By JC-1, propidium iodide and TUNEL and LDH release of CLN2-deficient human neurons (Dhar et al, 02).3. Resistance of stable CLN2-overexpressing NT2 cells to proapoptotic insults such as vincristine, staurosporine and etoposide (ibid).

4. Increased apoptotic death of CLN2-deficient, naturally occurring patient derived fibroblasts and lymphoblasts resulting in net slower growth (ibid).5. Restoration of growth by transfection of CLN2-deficient cells with a CLN2 bearing plasmid.

Apoptosis in NCLIn INCL:

Blocking of CLN1 expression results in apoptosis, and CLN1-overexpression protects from ceramide-induced cell death (Glyn Dawsons group)

Slowed growth of CLN1-deficient patient lymphoblasts and fibroblasts (unpublished)

Growth restored by transfection of CLN1-deficient fibroblasts with a CLN1-bearing plasmid (unpublished).

Chart1

11

2.71.8

3.72

42.3

4.72.7

53

CLN1-CLN1+

CLN1-/CLN1-

Time/hrs

cell count x 10^4

Growth curves of CLN1 deficient lymphoblasts

Sheet1

Time/hrsCLN1-/CLN3CLN1-CLN1+CLN3-CLN1+Time/hrsCLN1-CLN1+CLN1-/CLN1-

0111011

822182.71.8

221163.72

1443442.3

1.66666666672.66666666672404.72.7

161344453

231

253

1.66666666673.66666666672.6666666667Time/hrsCLN3-/CLN3+CLN3-CLN1+CLN3-/CLN3-

342420111

33482.421.5

153163.32.71.8

243343.532

40243404.23.52.5

453.7444.43.32.9

154

2.33333333334.66666666673.5666666667

44443

254

263

2.666666666753.3333333333

Sheet1

1

#REF!

CLN1-CLN1+

CLN1-/CLN1-

Time/hrs

Cell count x 10^4

Cross correction of CLN1 deficient cels with CLN3 cDNA

Sheet2

CLN3-/CLN3+

CLN3-CLN1+

CLN3-/CLN3-

Time/hrs

Cell count x 10^4

Cross correction of CLN3 deficient cells with CLN1 cDNA

Sheet3

CLN1-CLN1+

CLN1-/CLN1-

Time/hrs

cell count x 10^4

Growth curves of CLN1 deficient lymphoblasts

Chart1

77770.20.10.10.3

8.44.14.34.20.10.40.30.2

9.36.25.260.40.40.40.1

10.57.36.27.10.20.30.50.33

12.38.17.47.30.10.20.40.2

15.68.57.77.9

Normal

CLN3 def.

CLN1 def.

CLN2 def.

Time/hrs

cell count x 10^4

Comparative growth rates: CLN1-, CLN2-, CLN3-deficient and wild type lymphoblasts

Sheet1

Time/hrsNormalCLN3 def.CLN1 def.Time/hrsWTCLN3-/CLN3-CLN3-/CLN3+CLN1 def.

010.51055540

76.64030107.65.57.343

723860238.55.98.662

1044639341071077

1084.241.3333333333434711.277.276

96606059147.2791

966065

886260

2393.333333333360.666666666761.6666666667000

113.2106540.110.1440.12

9360980.1220.0990.21

11055800.2440.110.133

34105.473.666666666777.33333333330.120.080.19

13084630.210.10.08

1125680

1298885

47123.66666666677676

1328192

1428490

1968392

59156.666666666782.666666666791.3333333333

Time/hrsNormalCLN3 def.CLN1 def.CLN2 def.

07777

108.44.14.34.20.20.10.10.3

239.36.25.260.10.40.30.2

3410.57.36.27.10.40.40.40.1

4712.38.17.47.30.20.30.50.33

5915.68.57.77.90.10.20.40.2

Sheet1

000

0.1440.110.12

0.0990.1220.21

0.110.2440.133

0.080.120.19

0.10.210.08

WT

CLN3-/CLN3+

CLN3-/CLN3-

Time/ hrs

Cell count x10^4

Growth curve for normal vs NCL lymphoblasts

Sheet2

0.20.10.10.3

0.10.40.30.2

0.40.40.40.1

0.20.30.50.33

0.10.20.40.2

Normal

CLN3 def.

CLN1 def.

CLN2 def.

Time/hrs

cell count x 10^4

Comparative growth rates: CLN1-, CLN2-, CLN3-deficient and wild type lymphoblasts

Sheet3

Time/hrscontrolCLN2 transTime/hrscontrolCLN2 trans

00.10.100.10.1

16101.15.3

1.56232.38

2434210

101.55.3333333333473.59.3

285949

38

28

232.33333333338

28.5

211.5

210

34210

3.58

310

410

473.59.3333333333

610

39

311

5949

Sheet3

11

control

CLN2 trans

Time/hrs

Cell count x 10^4

Growth curves for CLN2 transfected fibroblasts

Sheet4

Sheet4

555161011

7.65.57.3

8.55.98.6

10710

11.277.2

147.27

WT

CLN3-/CLN3-

CLN3-/CLN3+

Time/ hrs

Cell count x10^4

5. Growth curve for normal vs NCL lymphoblasts

Sheet5

0.10.111

1.15.3

2.38

210

3.59.3

49

control

CLN2 trans

Time/hrs

Cell count x 10^4

6. Growth curves for CLN2 transfected fibroblasts

Sheet6

Sheet7

APOPTOSIS IN NCLIn CLN6 deficient vLINCL:

By EM and TUNEL of affected human brain (Lane et al. 96).Increased ceramide in vLINCL CLN6-deficient human brain and fibroblasts.By flow cytometry (sub-diploid peak), DNA fragmentation by gel electrophoresis, TUNEL staining of affected sheep brain and retina observed at 14 months of age (provided by Bob Jolly, Lane et al,).

Overexpression of CLN2 causes resistance to apoptosis

dJC-1 StainingV2 without etoposideS1C4 without etoposideV2 with etoposideS1C4 with etoposide

V2 without etoposideV2 with etoposideS1C4 without etoposideS1C4 with etoposidee PI Staining

Control-AAV2AS-CLN2 AAV2Transduction of post-mitotic human neurons with AS-CLN2 causes spontaneous neuronal apoptotic death and LDH release

Chart1

1100

0.2810.08750.00883883480.0125

0.456250.20.02651650430.0216506351

0.9541670.26250.08322910150.0433012702

Control-AAV2

AS-CLN2-AAV2

Time (hours)

cell number * 10,000

Sheet1

123AvgSt Dev

0 hrctl11110

cln211110

24 hrctl0.28750.2750.281250.0088388348

cln20.0750.08750.10.08750.0125

48 hrctl0.43750.4750.456250.0265165043

cln20.18750.2250.18750.20.0216506351

72 hrctl0.86251.0250.9750.95416666670.0832291015

cln20.31250.23750.23750.26250.0433012702

Control-AAV2AS-CLN2-AAV2

011

240.2810.0875

480.456250.2

720.9541670.2625

Sheet1

0000

000.00883883480.0125

000.02651650430.0216506351

000.08322910150.0433012702

Control-AAV2

AS-CLN2-AAV2

Time (hours)

cell number * 10,000

Sheet2

Sheet3

Chart3

28.8

43.3

LDH release (% of Total)

Sheet4

Sheet5

Sheet1


28.843.3

Sheet1


Sheet2

Sheet3

MBD0000CE6E.xls

Chart1

28.8

43.3


Sheet4

Sheet5

Sheet1


28.843.3

Sheet1

0

0


Sheet2

Sheet3

Transduction of NT2 precursor cells with antisense CLN3 adenovirus causes cell death and LDH release

Chart2

555000

6.645.684.20.450.370.05

9.369.411.920.720.760

17.6418.271.30.840.680.17

23.3622.10.251.470.490.014

w/o

cont

Ad-AS CLN3

Time (hours)

cell number (*10,000)

Sheet1

0point55

w/o182111171215.86.326.64

121722201617.46.960.45254834

171710913.255.3

cont virus 15MOI1518132271565.68

131313161714.45.760.3666060556

71914121413.25.28

Ad-AS CLN3 15MOI776476.22.484.2

8101571310.64.240.0565685425

10111211810.44.16

w/o271921212021.68.649.36

302423221823.49.360.72

301929212725.210.08

cont virus242532212625.610.249.4133333333

171920332723.29.280.768721883

231619302121.88.72

Ad-AS CLN3463294.81.921.92

3124414.81.920

244523.41.36

35hours

w/o3835394539.2515.717.64

413954374242.617.040.8485281374

394544505045.618.24

cont virus424741436447.418.9618.2666666667

494941474245.618.240.6803920438

43484342444417.6

Ad-AS CLN3723433.81.521.3333333333

253423.21.280.16653328

2552131.2

47hours0

w/o435464665255.822.3223.36

393641523640.816.321.4707821049

56557360616124.4

cont virus695661415656.622.6422.1066666667

416361545655220.4888080741

525159604954.221.68

Ad-AS CLN3621312.60.260.25

415684.80.240.0141421356

Sheet2

011233547

w/o56.649.3617.6423.3600.450.720.841.47

cont55.689.4118.2722.100.370.760.680.49

Ad-AS CLN354.21.921.30.2500.0500.170.014

Sheet2

000

0.450.370.05

0.720.760

0.840.680.17

1.470.490.014

w/o

cont

Ad-AS CLN3

Time (hours)

cell number (*10,000)

Sheet3

Sheet4

Sheet5

Sheet6

Sheet7

Sheet8

Chart1

25.472270.881055

47.15061.099515

LDH release (% of total)

Sheet1

Ad-ControlAd-AS-CLN3

25.4722747.1506

Sheet1

0

0


LDH release from hNT neurons after transduction Ad-AS CLN3 virus and control virus

Sheet2

0.8810551.099515

Ad-controlAd-AS-CLN3

Sheet2

25.472270.881055

47.15061.099515


Sheet3

Sheet4

Sheet5

Sheet6

Sheet7

Sheet8

Ad-control (without etoposide)Ad-control (with etoposide)Ad-AS-CLN3 (without etoposide) Ad-AS-CLN3 (with etoposide)Transduction of hNT neurons with AS-Ad-CLN3 results in spontaneous apoptotic death by TUNEL staining

FlupirtineIt is a triaminopyridine analogue. It was developed and is used as a non-opioid centrally acting analgesic.It has powerful muscle-relaxant properties not mediated through the benzodiazepine receptor (ideal for treatment of back pain with spasms, orthopedic and cancer pain).It has been shown to protect postmitotic neurons and photoreceptors from apoptotic death induced by a variety of insults: NMDA, amyloid, HIV and prionic proteins. It has been shown to reduce the area of infarction in rats, and to improve their motor function following carotid artery ligation. It has been shown to elevate Bcl-2 levels 5-7 fold, and glutathione levels.There is an indirect functional antagonism to NMDA-induced cell death.It activates a G-regulated, inwardly rectifying K+ channel.It stabilizes retention of Ca++ at the mitochondrial membrane level.

Beneficial Clinical Characteristics of Flupirtine Can be given orally/per gastrostomy tubeProven safety after prolonged use in adults and children (for periods > 2years). Has been in use for 18 years.*Pain-killer, less sedating than opioids.* Muscle relaxant properties.Centrally acting (ideal, as this is where you want it to act).* Weak anti-convulsant action, and very little interaction with other anticonvulsants.Cheap. *Pain, spasticity, and seizures are key target symptoms in NCL

Normal Controlmno

Flupirtine Protects CLN1-deficient lymphoblasts from apoptosisacb

Flupirtine protects CLN2-deficient lymphoblasts from etoposide-induced apoptosisedf

Flupirtine protects CLN3-deficient lymphoblasts from etoposide induced apoptosisigh

Flupirtine protects CLN6-deficient lymphoblasts from etoposide-induced apoptosisljk

Flupirtine protects healthy hNT neurons from etoposide induced apoptosis cab

Flupirtine protects hNT neurons transduced with AS-CLN2-AAV2 from apoptosisFlupirtine protects hNT neurons transduced with Ad-AS-CLN3 from apoptosisighij+ Flup+ Flup

FlupirtineIn Vitro Experimentation: DonePre-IND Application and FDA meeting : DonePhase I/II Pilot Clinical Trial in LINCL NIH application to be resubmitted. Why LINCL first? Can determine within shorter period than for JNCL whether effective. INCL fewer patients, and other oral therapies available. Once FDA approved can be used off-label for other indications.IND Application: To be submitted as soon as grant resubmitted.

COLLABORATORS: VIATRIS (drug); GENZYME (regulatory support); BDSRA (financial and logistical support); NINDS: G. Spinella, Dan Tagle and D. Hirtz (advice on design and strategy); DCRI (statistics and clinical trial expertise), A. Kohlschtter (clinical endpoints) IBR: K. Wisniewski & N. Zhong; MGH: Katherine Sims; Toronto Sick Kids: B. Minassian; BDSRA: Lance Johnston (diagnosis and patient referral).

Co-workers and Collaborators

Co-Workers (Batten Brigade)Present: Dixie-Ann Persaud SawinAngela SchulzAlisa RayGabreal St.ClairePrevious: Sumeer DharSvetlana RylovaRhonda BittingPaul JansenEllen LockhartCarla Andreia Teixeira

CollaboratorsAndrea AmalfitanoDwight KoeberlAlfried Kohlschtter

ACKNOWLEDGEMENTSFamilies and affected children, BDSRA, NINDS, Battin for Betsy Charities, Inc., Serbian Orthodox Church, McGee FamilySandy Hoffman (CLN1 cells), P. Lobel and D. Sleat (CLN2 cDNA), Nan Zhong, VIATRIS, and Genzyme

Documents

Use of the oral antiapoptotic agent Flupirtine as a treatment strategy in human INCL, LINCL, JNCL and CLN6-deficient vLINCL