Drug discovery to Market, Sitagliptin case study, Presented at At H( S ) NCB 's Dr L H Hiranandani college of pharmacy , ulhasnagar, India for key note speech in a one day symposium on APi synthesis from lead optimisation to patent filing 17 dec 2016...Dept of Pharmaceutical chemistry

1. A JOURNEY FROM DRUG
DISCOVERY TO MARKET,
SITAGLIPTIN CASE STUDY
SPECIALLY FOR
SEMINAR AT DR. L. H. HIRANANDANI COLLEGE OF PHARMACY, DEPT OF
PHARMACEUTICAL CHEMISTRY, DLHHCOP, ULHASNAGAR, MAHARASHTRA, INDIA
A JOURNEY FROM DRUG
DISCOVERY TO MARKET,
SITAGLIPTIN CASE STUDY
SPECIALLY FOR
SEMINAR AT DR. L. H. HIRANANDANI COLLEGE OF PHARMACY, DEPT OF
PHARMACEUTICAL CHEMISTRY, DLHHCOP, ULHASNAGAR, MAHARASHTRA, INDIA
DR ANTHONY MELVIN CRASTO
PRINCIPAL SCIENTIST
WORLDDRUGTRACKER
17 DEC, 2016
1

2. 2
" API- From lead optimization
to patent filing".

3. 3
A National level seminar on 17th December, 2016.
The Theme for the seminar is " API- From Lead
optimization to Patent filing" at Dr. L. H. Hiranandani
College of Pharmacy, Dept of pharmaceutical chemistry,
DLHHCOP, Ulhasnagar, Maharashtra, India

4. 4
Anita Ayre
Assistant Professor at
Dr.L.H.Hiranandani College of Pharmacy

5. THANKS
TO
VEDIKA
DADLANI,
KUNAL GOKHALE
AND
ANITA
AYRE
Good
planning
5

6. PRINCIPAL AT
DR.L.H.HIRANANDANI COLLEGE
OF PHAR
Thanks
6

7. 7

8. W
HY
I CHOSE
SITAGLIPTIN
AS
EXAM
PLE
SEE
NEXT
SLIDE
8

9. GODDESS
SITA
FROM
RAM
AYAN
Our
role
m
odel JAI SRI RAM
, and
SITA
M
AIYA
9

10. 10
RAMIPRIL, has LORD RAM NAME IN THE NAME

11. STRUCTURE
11

12. 12
Januvia (Sitagliptin Phosphate) Tablets
Company: Merck & Co., Inc.
Application No.: 021995
Approval Date: 10/16/2006
Approval Letter(s) (PDF)
Printed Labeling (PDF)
Medical Review(s) (PDF)
Chemistry Review(s) (PDF)
Pharmacology Review(s) (PDF)
Statistical Review(s) (PDF)
Clinical Pharmacology Biopharmaceutics Review(s)
(PDF)
Administrative Document(s) & Correspondence
(PDF)

13. 13

14. 14
• In October 2006, the U.S. Food and Drug Administration
(FDA) approved Sitagliptin as monotherapy and as add-on
therapy to either of two other types of oral diabetes
medications.
• In April, 2007 FDA approved the combination product of
Sitagliptin and Metformin for type 2 diabetes.
• In March, 2007 it was approved in European Union.
• Sitagliptin is currently approved in 70 Countries.
Regulatory Affairs

15. 15

16. 16

17. Dipeptidyl Peptidase 4 (DPP-4)
Inactivates GLP-1
Mixed meal
Intestinal
GLP-1
release
Rapid inactivation
Excreted by kidneys
GLP-1
Active
DPP-4
GLP-1 Actions
GLP-1
Inactive
Diabetes.1995;44:1126
Clinical Therapeutics.2006;28(1):55
Pharmacology & Therapeutics.2010:125;328–361

18. Drug Review.2008;10(2):97-98
• Reduces hemoglobin A1c (HbA1c), fasting and
postprandial glucose by glucose dependant stimulation of
insulin secretion and inhibition of glucagon secretion.
• Sitagliptin is selective inhibitor of the enzyme DPP-4.
• Delays gastric emptying and reduce appetite.
18
SITAGLIPTIN
18
Mechanism of action (MOA)

19. 19
Sitagliptin is a triazolopiperazine based inhibitor of DPP-IV,
which was discovered byMerck. It is a potent (IC50= 18 nM)
and highly selective over DPP-8 (48000 nM), DPP-9
(>100000 nM) and other isozymes. It enhances the
pancreatic β-cell functions, fasting and post-prandial
glycemic control in type 2 diabetic patients. In the crystal
structure with DPP-IV, unlike other substrate-based DPP-IV
inhibitors, the binding orientation of the amide carbonyl of
sitagliptin is reversed, i.e. the aromatic trifluorophenyl moiety
occupies S1 pocket and the β-amino amide moiety fits into
S2 pockets. The amino group forms a salt bridge and
hydrogen bonding interactions with Glu205 and Glu206, and
Tyr662, respectively.
The triazolopiperazinemoiety occupies the S2 extended
pocket and stacks against Phe357.

20. 20
The exhibited binding interactions of the trifluoromethyl
group with the Arg358 and Ser209 are responsible for
its high selectivity profile. The presence of the
trifluoromethyl group in the triazole ring also
improvesthe oral bioavailability in animal models.
Sitagliptin inhibited the plasma DPP-IV up to 80% and
47% at 2 and 24 h, respectively, after a single dose of
25.0 mg in a dose-dependent manner. In a 24-week
study, sitagliptin significantly decreased fasting
glucose levels and HbA1c levels (0.8%) at doses of
100 mg q.d. Thus, sitagliptin is well tolerated and body
weight neutral. It is the first DPP-IV inhibitor in the
class approved by USFDA in 2006 and is used as
either a monotherapy or in combination with metformin

21. 21

22. 22
http://www.druglead.com/cds/sitagliptin.html
Title: Sitagliptin
CAS Registry Number: 486460-32-6
CAS Name: 7-[(3R)-3-Amino-1-oxo-4-(2,4,5-trifluorophenyl)butyl]-5,6,7,8-
tetrahydro-3-(trifluoromethyl)-1,2,4-triazolo[4,3-a]pyrazine
Additional Names: (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-
dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-
amine
Molecular Formula: C16H15F6N5O
Molecular Weight: 407.31
Percent Composition: C 47.18%, H 3.71%, F 27.99%, N 17.19%, O 3.93%
Literature References: Selective inhibitor of dipeptidyl peptidase IV (DPP-IV).
Prepn: S. D. Edmondson et al., WO 03004498; eidem, US 6699871 (2003, 2004
both to Merck & Co.); and enzyme inhibitory profile: D. Kim et al., J. Med.
Chem. 48, 141 (2005). Improved process: K. B. Hansen et al., Org. Process Res.
Dev. 9, 634 (2005). Clinical pharmacokinetics and pharmacodynamics: G. A.
Herman et al., Clin. Pharmacol. Ther. 78, 675 (2005). Review: C. F.
Deacon, Curr. Opin. Invest. Drugs 6, 419-426 (2005).
Properties: Viscous oil.

23. 23
Derivative Type: Monophosphate monohydrate
CAS Registry Number: 654671-77-9; 654671-78-0 (anhydrous)
Additional Names: Sitagliptin phosphate
Manufacturers' Codes: MK-0431
Trademarks: Januvia (Merck & Co.)
Molecular Formula: C16H15F6N5O.H3PO4.H2O
Molecular Weight: 523.32
Percent Composition: C 36.72%, H 3.85%, F 21.78%, N 13.38%, O 18.34%,
P 5.92%
Properties: mp 215-217°. [a]D -74.4° (c = 1.0 in water).
Melting point: mp 215-217°
Optical Rotation: [a]D -74.4° (c = 1.0 in water)

24. 24
Orange book
NDA 021995
JANUVIA (SITAGLIPTIN PHOSPHATE)
EQ 25MG BASE
Active Ingredient: SITAGLIPTIN PHOSPHATE
Proprietary Name: JANUVIA
Dosage Form; Route of Administration: TABLET; ORAL
Strength: EQ 25MG BASE
Reference Listed Drug: No
TE Code:
Application Number: N021995
Product Number: 001
Approval Date: Oct 16, 2006
Applicant Holder Full Name: MERCK SHARP AND DOHME CORP
Marketing Status: Prescription
Patent and Exclusivity Information
http://www.accessdata.fda.gov/scripts/cder/ob/

25. 25

26. 26
Product No Patent No
Patent
Expiration
Drug
Substance
Claim
Drug Product
Claim
Patent Use
Code
Delist
Requested
001 6699871 Jul 26, 2022 DS DP U-774
001 7125873 Jul 26, 2022
U-775 U-1036
U-1037 U-1038
001 7326708 Nov 24, 2026 DS DP U-802

27. 27
Patent Data
U-774
METHOD OF TREATING TYPE 2 DIABETES MELLITUS BY
ADMINISTERING A DIPEPTIDYL PEPTIDASTE-IV INHIBITOR
US 7125873
http://www.google.co.in/patents/US7125873
CA2450740A1, CA2450740C, CN1290848C, CN1524082A, CN1861077A,
DE60210093D1, DE60210093T2, DE60236767D1, DE122007000056I1, DE122008000046I1,
EP1412357A1, EP1412357B1, EP1625847A1, EP1625847B1, EP2226324A1, EP2292232A1,
EP2292232B1, US6699871, US8168637, US8440668, US20030100563, US20040167133,
US20060270679, US20100130504, US20110190308, US20130217695, US20150359793,
WO2003004498A1

28. 28

29. 29
NDA CHEMISTRY REVIEWS
http://www.accessdata.fda.gov/drugsatfda_docs/nda/2006/021995s000_Chem
R.pdf

30. 30

31. 31

32. DISCOVERY SYNTHESIS
PATENT WO2003004498
SCOTT D EDMONDSON,
MICHAEL H FISHER, DOOSEOP KIM,
MALCOLM MACCOSS, EMMA R PARMEE,
ANN E WEBER, JINYOU XU
32Emma Parmee
Ann E Weber
Scott D. Edmondson

33. 33

34. 34
A new synthesis of sitagliptin (MK-0431), a DPP-IV inhibitor and potential
new treatment for type II diabetes, suitable for the preparation of multi-
kilogram quantities is presented. The triazolopyrazine fragment of
sitagliptin was prepared in 26% yield over four chemical steps using a
synthetic strategy similar to the medicinal chemistry synthesis. Key
process developments were made in the first step of this sequence, the
addition of hydrazine to chloropyrazine, to ensure its safe operation on a
large scale. The beta-amino acid fragment of sitagliptin was prepared by
asymmetric reduction of the corresponding beta-ketoester followed by a
two-step elaboration to an N-benzyloxy beta-lactam. Hydrolysis of the
lactam followed by direct coupling to the triazolopiperazine afforded
sitagliptin after cleavage of the N-benzyloxy group and salt formation.
The overall yield was 52% over eight steps.

35. 35

36. 36

37. 37
The synthesis of 1 was completed using a four-step
through-process
Lactam 5 or ester 13 was hydrolyzed to amino acid 2b with
LiOH in THF/water by either stirring at room temperature
or, in the case of 13, heating to 40 °C.
While the benzyloxy group of 2b could be cleaved by
hydrogenacoupling to triazole 3, the benzyloxy group of 2b
was found to sufficiently tion and then protected with Boc2O
to prevent side reactions during the protect the amino
group to allow the desired amide to be formed.
Thus, triazole 3 was coupled to 2b at 0 °C using EDC−HCl
and N-methylmorpholine (NMM) as base to afford 14 in
>99% assay yield.

38. 38
Following an aqueous workup, the organic extracts were distilled
into ethanol and the solution was subjected to hydrogenation with
10% Pd on carbon.
The presence of water in the hydrogenation was found to be crucial
to the reaction success; anhydrous solutions of 14 hydrogenated
with dry Pd on carbon proceeded only to low levels of conversion to
1, and addition of water to these reductions resulted in restored
performance of the catalyst.
Following hydrogenation, the catalyst was removed by filtration to
provide an ethanol solution of 1.
Sitagliptin was isolated in >99.5% purity as its anhydrous
phosphoric acid salt by crystallizing from aqueous ethanol.

39. 39

40. SYNTHESIS 2
COMPD NUMBERING DIFFERENT
40

41. 41

42. 42
The second approach for synthesis of sitagliptin was
started from asymmetric reduction of β-ketoester 15 using
the (S)-BinapRuCl2 complex with a catalytic amount of
HBr in methanol
followed by hydrolysis afforded the β-hydroxy acid 16.
Lactam 17 was synthesized by coupling of 16 with
BnONH2 •HCl using N-(3- dimethylaminopropyl)-N-
ethylcarbodiimide hydrochloride (EDC),
Followed by cyclization reaction with diisopropyl
azodicarboxylate (DIAD) and PPh3 .

43. 43
Treatment of a catalytic amount of 0.1% NaOH with lactam
17 hydrolyzed and directly afforded the β-amino acid 18.
This wascoupled withtriazolopiperazine 5 using EDC•HCl
and N-ethylmorpholine to provide the N-benzyloxy protected
compound 19,
Which after hydrogenation using Pd/C and by consequent
treatment with phosphoric acid provided the phosphate salt
of sitagliptin

44. 44

45. 45
The third approach towards the synthesis of sitagliptin is
outlined in scheme 3.
Meldrum adduct 22 (Hunig’s base salt) was synthesized from
trifluorophenylacetic acid 20 by the formation of a mixed
anhydride with pivaloyl chloride in the presence of Meldrum’s
acid 21, DIPEA and catalytic amount of dimethylamino
pyridine (DMAP) in acetonitrile.
Treatment of 22 with TFA resulted compound 23. β-keto
amide 24 was formed on reaction of 23 with
triazolopiperazine 5

46. 46
β-keto amide 24 was formed on reaction of 23 with
triazolopiperazine 5.
β-keto amide 24 on treatment with ammonium acetate in
methanol formed a key intermediate, dehydrositagliptin
25 (enamine amide).
This intermediate contains the entire structure of
sitagliptin 14 except two hydrogen atoms.
Thus, sitagliptin 14 was synthesized by enantioselective
hydrogenation of dehydrositagliptin 25 in the presence of
[Rh(COD)2 OTf] and t Bu JOSIPHOS in excellent yield
with 95% ee.

47. 47

48. OMARIGLIPTIN VS SITAGLIPTIN
48
ONCE A DAY SITAGLIPTIN
For patients with moderate renal insufficiency (CrCl greater than or equal to 30 to
less than 50 mL/min, approximately corresponding to serum creatinine levels of
greater than 1.7 to less than or equal to 3.0 mg/dL in men and greater than 1.5 to
less than or equal to 2.5 mg/dL in women), the dose of JANUVIA is 50 mg once
daily.
MARIZEV® (Omarigliptin), Merck’s Once-Weekly DPP-4 Inhibitor for
Type 2 Diabetes, Approved in Japan
-Merck, known as MSD outside the United States and Canada, today
announced that the Japanese Pharmaceuticals and Medical Devices
Agency (PMDA) has approved MARIZEV®
(omarigliptin) 25 mg and 12.5
mg tablets, an oral, once-weekly DPP-4 inhibitor indicated for the
treatment of adults with type 2 diabetes. Japan is the first country to have
approved omarigliptin.

49. 49

50. 50

51. 51
Omarigliptin (MK-3102, Merck) Omarigliptin is a novel
aminotetrahydropyran based structurally distinct rigid analogue of
sitagliptin in which the central linker of sitagliptin was modified to rigid
cyclohexylamine. It is extremely potent (IC50=1.6 nM) and long acting
with excellent selectivity profile against isopeptidases. It is once-a-week
oral agent rather than once a day like existing therapies in DPP-IV
inhibitor. Omarigliptin binds in the DPP-IV active site very similar to
sitagliptin and share the same interactions. The 2-F atom in the
trifluorophenyl group occupied in S1 pocket shows a hydrogen bonding
with the side chain of Arg125. On the other hand, the
aminotetrahydropyran group binds to S2 pocket where the primary
amine group makes salt bridges with the carboxylates of Glu205 and
Glu206. The fused ring π-π stacks with the side chain of Phe347.[65]
Omarigliptin exhibited a unique PK and pharmacological profile with the
half-life of ~68 h. It markedly lowered the blood glucose levels and
0.57% of HbA1c level at a dose of 25 mg in a 12 week study. It also
exhibited 77- 89% inhibition of plasma DPP-IV up to 168 h and
enhanced two folds of GLP-1 levels. Omarigliptin is generally well
tolerated with excellent safety profiles in healthy subjects. Currently,
omarigliptin is in clinical trial PhaseIII

52. 52
https://newdrugapprovals.org/
https://newdrugapprovals.org/2014/04/18/o
marigliptin-mk-3102-in-phase-3-for-type-2-
diabetes/

53. 53

54. Pharmacokinetics
 Bioavailability of Sitagliptin is approximately 87% .
 Half life is between 8-14 hours.
 It is 38% bound to plasma proteins.
 Elimination is mainly through urine.
Drug Review.2008;10(2):97-9854

55. 55
Sitagliptin (Januvia) has a novel structure with β-amino amide derivatives
Since sitagliptin has shown excellent selectivity and in vivo efficacy it
urged researches to inspect the new structure of DPP-4 inhibitors with
appended β-amino acid moiety.
Further studies are being developed to optimize these compounds for the
treatment of diabetes.
In October 2006 sitagliptin became the first DPP-4 inhibitor that got FDA
approval for the treatment of type 2 diabetes. Crystallographic structure
of sitagliptin along with molecular modeling has been used to continue
the search for structurally diverse inhibitors.
A new potent, selective and orally bioavailable DPP-4 inhibitor was
discovered by replacing the central cyclohexylamine in sitagliptin with 3-
aminopiperidine.
A 2-pyridyl substitution was the initial SAR breakthrough since that group
plays a significant role in potency and selectivity for DPP-4.

56. 56
It has been shown with an X-ray crystallography how
sitagliptin binds to the DPP-4 complex:
1.The trifluorophenyl group occupies the S1-pocket
2. The trifluoromethyl group interacts with the side
chains of residues Arg358 and Ser209.
3. The amino group forms a salt bridge with Tyr662
and the carboxylated groups of the two glutamate
residues, Glu205 and Glu206.
4. The triazolopiperazine group collides with the
phenyl group of residue Phe357

57. 57
a) reducing both fasting and postprandial glucose
concentration,
b) clinically meaningful reductions in glycosylated
hemoglobin (HbA1c) levels in type 2 diabetic patients.
• Monotherapy with Sitagliptin 100 mg daily decreases mean
HbA1c by 0.6-0.98%.
CLINICAL EVIDENCE
Drug Review.2008;10(2):97-98
Consultant.2009:S5-11
Pharmacology & Therapeutics.2010;25:328-361
• In very well controlled randomized clinical trials Sitagliptin
(100 mg) treatment significantly improved glycemic control
by
• Improved Homeostasis model assessment of β cell and
Proinsulin-to-insulin ratio.

58. 58
• The recommended dose of Sitagliptin is 100 mg once
daily. It may be taken with or without food.
Recommended Dosage

59. 59
Marketed Brands
 Januvia (Sitagliptin)
 Janumet (Sitagliptin and Metformin)
9323115463 amcrasto@gmail.com

60. 60
Summary of Sitagliptin
 No clinically meaningful hypoglycemia
 Weight neutral
 DPP-4 Inhibitor
 Good tolerability
 Improves Blood pressure
 Stimulate insulin secretion
 Slows gastric emptying
 Reduces food intake
 Inhibit glucagon secretion
 Reduces HbA1c
 Improves inflammatory markers

61. CLINICAL TRIALS
61

62. 62
https://clinicaltrials.gov/search/intervention
=sitagliptin+phosphate

63. 63

64. 64
http://www.centerwatch.com/drug-information/fda-approved-
drugs/drug/935/januvia-sitagliptin-phosphate#

65. 65

66. 66

67. 67

68. 68
GREEN CHEMISTRY

69. 69https://orgspectroscopyint.blogspot.in/2015/03/sitagliptin.html

70. 70
Recently, large pharmaceutical process chemists have relied heavily on the
development of enzymatic reactions to produce important chiral building blocks for
API synthesis. Many varied classes of naturally occurring enzymes have been co-
opted and engineered for process pharmaceutical chemistry applications.
The widest range of applications come from ketoreductases and transaminases, but
there are isolated examples from hydrolases, aldolases, oxidative enzymes,
esterases and dehalogenases, among others.[11]
One of the most prominent uses of biocatalysis in process chemistry today is in the
synthesis of Januvia®, a DPP-4 inhibitor developed by Merck for the management
of type II diabetes. The traditional process synthetic route involved a late-stage
enamine formation followed by rhodium-catalyzed asymmetric hydrogenation to
afford the API sitagliptin.
This process suffered from a number of limitations, including the need to run the
reaction under a high-pressure hydrogen environment, the high cost of a transition-
metal catalyst, the difficult process of carbon treatment to remove trace amounts of
catalyst and insufficient stereoselectivity, requiring a subsequent recrystallization
step before final salt formation.[12][13]

71. 71
Merck’s process chemistry department contracted Codexis, a medium-sized
biocatalysis firm, to develop a large-scale biocatalytic reductive amination for
the final step of its sitagliptin synthesis. Codexis engineered a transaminase
enzyme from the bacteria Arthrobacter through 11 rounds of directed
evolution.
The engineered transaminase contained 27 individual point mutations and
displayed activity four orders of magnitude greater than the parent enzyme.
Additionally, the enzyme was engineered to handle high substrate
concentrations (100 g/L) and to tolerate the organic solvents, reagents and
byproducts of the transamination reaction.
This biocatalytic route successfully avoided the limitations of the
chemocatalyzed hydrogenation route: the requirements to run the reaction
under high pressure, to remove excess catalyst by carbon treatment and to
recrystallize the product due to insufficient enantioselectivity were obviated by
the use of a biocatalyst.
Merck and Codexis were awarded the
Presidential Green Chemistry Challenge Award in 2010 for the development of
this biocatalytic route toward Januvia®.[14]

72. 72
GREENING UP DRUGS Merck process chemists redesigned and significantly
shortened the original synthesis of type 2 diabetes drug candidate sitagliptin (Januvia)
to include an unprecedented efficient hydrogenation of an unprotected enamine.
Chemical & Engineering News • ISSN 0009-2347 • Copyright © 2006 American Chemical Society

73. 73

74. SOM
E
CLIPS
74

75. 75
Despite this process being a considerable improvement over its predecessor,
the late-stage hydrogenation was only moderately stereoselective and required
high-pressure conditions [151].
The removal of the metal catalyst by absorption onto a polymer impregnated
with activated carbon and the final recrystallization as the [H2PO4]−
salt led to
reduced yield [151].

76. 76
Enzymatic synthesis of sitagliptin [152].
http://rspa.royalsocietypublishing.org/content/471/2183/20150502

77. 77
The final version of the sitagliptin synthesis avoided this
hydrogenation by using a transaminase enzyme to directly
aminate the prositagliptin diketone precursor with iso-
propylamine (scheme 10) [152], giving a highly enantiopure
product.
The enzymatic process gives a 10–13% increase in overall
yield, a 53% increase in productivity (kgl −1
day −1
), a 19%
reduction in total waste and the elimination of all heavy
metals.
In addition to these environmental advantages, the
biocatalytic process eliminated the need for specialized
high-pressure equipment, leading to reductions in both
capital and running costs.

78. DMF

79. 79

80. 80
http://www.pharmacompass.com/us-drug-master-files-dmfs/sitagliptin-
phosphate

81. 81
http://www.usactives.com/z1/search.php?
zoom_query=sitagliptin&zoom_per_page=10&zoom_and=0&zoom_sort=0

82. 82
http://www.pharmacompass.com/pharma/ingredients/Sitagliptin_Phosphate

83. FORMULATIONS

84. 84

85. 85
http://www.rxlist.com/janumet-xr-drug.htm

86. ANALYSIS

87. 87
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-
46702010000300003

88. New Drug Approvals
https://newdrugapprovals.org/

89. MY BLOGS
ORGANIC SPECTROSCOPY INTERNATIONAL
LINK….. http://orgspectroscopyint.blogspot.in/
ORGANIC SPECTROSCOPY INTERNATIONAL
Organic Chemists from Industry and academics to Interact on
Spectroscopy Techniques for Organic Compounds ie NMR, MASS,
IR, UV Etc. Starters, Learners, advanced, all alike, contains content
which is basic or advanced, by Dr Anthony Melvin Crasto,
Worlddrugtracker, email me ........... amcrasto@gmail.com, call +91
9323115463

90. LIONEL MY SON
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was hit by a deadly one in a million spine
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He cried bitterly and we had never seen him
so depressed
Now I keep Lionel as my source of inspiration
and helping millions, thanks to millions of my
readers who keep me going and help me to
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91. To take full advantage of Chemical Web content, it is essential
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92. SUCCESS FORMULA

93. 93THANK YOUTHANK YOU