1. P. S. KUNDHAL ET AL. JAMA November, 2013 Moderator – Dr Vineet Ahuja
Effect of Thalidomide on clinical remission in children and adolescents with refractory Crohn’s disease
P. S. KUNDHAL ET AL.
JAMA November, 2013
Moderator – Dr Vineet Ahuja

2. Introduction
1.2 million people in Europe and more than half million in the United States are estimated to have Crohn’s disease
Prevalence is particularly high in North America and Europe (319 to 322 per )
The highest annual incidence of CD was 12.7 per 100,000 person-years in Europe, 5.0 person-years in Asia and the Middle East, and 20.2 per 100,000 person-years in North America.
Molodecky NA, Gastroenterology 2012

3. 25% people of CD develop symptoms as children
Resistance or intolerance to therapy is common in children
Approximately 18%of cases require surgery within 5 years from onset
If not adequately treated, children with Crohn disease may have permanent impairments

4. Thalidomide is a small molecule with anti TNF α, immunomodulatory, and antiangiogenic properties
Observational studies on thalidomide have reported encouraging results, with remission rates ranging from 40% to 70%
Laffitte E, Expert Opin Drug Saf,2004
Ginsburg PM, Ann Med,2001

5. Aim
To determine whether thalidomide is effective in inducing remission in refractory pediatric crohn’s disease

6. PCDAI score ranges from 0 to 95 score >10 indicating active disease
Clinical activity was measured by the Pediatric Crohn Disease Activity Index (PCDAI)
PCDAI score ranges from 0 to 95
score >10 indicating active disease
Score ≥ 30 indicate moderate to severe disease
Score ≥ 15 required for inclusion

7. Resistance to immunosuppressants was defined as active disease despite
Prednisone, 2mg/kg per day (maximum 60 mg/d) or the equivalent for 8 weeks
Azathioprine or mercaptopurine for 4 months
Methotrexate for 3 months
Infliximab 5 mg/kg at 0, 2, and 6 weeks
Cyclosporine, oral 2 mg/kg per day for 4 weeks or intravenously 1 mg/kg per day for 1 week

8. Inclusion Criteria Children and adolescents aged 2 to18 yrs
Active crohn’s disease despite immunosuppresion
Adverse events preventing continuous treatment

9. Exclusion criteria Requiring immediate surgery Ongoing pregnancy
Neuropathy
HIV /Tumors
Transplanted organs
ongoing major infections
Participation in other experimental studies,
Infliximab in the previous 8 weeks

10. Study Design Multicenter, Double-blind,placebo-controlled, RCT (Italy)
Randomised to thalidomide or placebo and followed for 8 weeks
In placebo group who at 8 weeks were not in clinical remission or did not have 75% response were given thalidomide
Followed up in an open-label extension for an additional 8 weeks to verify whether they responded to thalidomide after failure with placebo
After RCT phase, responders to thalidomide were followed up to additional 52 weeks to document long-term efficacy and adverse events related to thalidomide

11. Randomization and Masking
Randomization done by computer generated list
Both clinicians and children and their families were blinded for the randomized controlled trial phase (8 weeks)
Subsequently, the study continued open label

12. Study Treatment
Thalidomide given in dosage of 50,100,or 150 mg to patients weighing <30 kg, 30 to 60 kg, and >60kg,respectively
Immunosuppressant use was suspended except tapering steroids because of a relapse

13. Evaluation of Efficacy and Adverse Events
Done weeks 0, 4 and 8
Diary data
General patient condition,
Frequency and type of abdominal pain
Stool characteristics
Any other complaint .Laboratory samples
Hematocrit
Ferritin
ESR
CRP
Albumin
Electrolytes
PCDAI and nutritional indicators were calculated, and adverse
events were recorded

14. Adverse events Evaluation conducted at each visit A detailed history
Vital signs
Physical examination
Laboratory analysis

15. Peripheral neuropathy is a possible adverse event related to use of Thalidomide
Electromyography (EMG) was performed at weeks 8 to 12 for all patients
With peripheral neuropathy had to cease receiving thalidomide

16. Primary End Points
Clinical remission at week 8, defined by a PCDAI score of 10 or less
Reduction in PCDAI score of ≥ 25% or ≥ 75%, measured at weeks 4 and 8

17. Secondary End Points Mean PCDAI score CRP level ESR BMI Weight for age
Physician Global Assessment score
Incidence of adverse effects

18. Longer-term Follow-up
Responders to thalidomide were followed prospectively for a minimum of 52 weeks
Outcomes were evaluated at 12, 16, 26, and 52 weeks and every 26 weeks thereafter
Primary efficacy outcomes were clinical remission and clinical response 75%
Secondary outcomes included mean time to reach remission, mean PCDAI score, steroid suspension, and thalidomide dose
Adverse events were monitored at each visit, and EMGs were repeated every 3 months during the follow-up

19. Statistical Analysis
Results from the group randomized to thalidomide were compared with those of the group who later began receiving thalidomide, with statistical tests for unpaired data,
Results from the group who later began receiving thalidomide were compared with those from the placebo group, using tests for paired data.

20. Multivariable logistic regression analysis used to evaluate if baseline characteristics were associated with the primary outcome (clinical remission)
Maintenance of clinical remission over time analyzed by using Kaplan-Meier curves, and the log- rank test was used to compare differences between them.
Adverse events were reported as incidence for the randomized controlled trial phase and as cumulative incidence (number of events/patient-weeks) for the long-term follow-up

21. Flow chart of patient in trial

22. Baseline characteristics were similar among groups, except for the mean ESR value, which was higher in patients randomized to thalidomide

23. Baseline charcterstics

24. Baseline characterstics

25. Results Primary End Point At week 8,
13 of 28 children receiving thalidomide (46.4%)
compared with 3 of 26 of those receiving
placebo (11.5%) reached clinical remission
(P<.01)

26. At 4 weeks
25% and 75% responses were not different
between groups
At week 8,
Thalidomide group had a better response
25% response % vs 30.8%
75% response % vs 11.5%
( P <.01 for both)

27. PCDAI scores of groups by time

28. Secondary end points At week 8,
Mean PCDAI score, ESR, weight for age, and physician global
assessment scores were all significantly improved in the
thalidomide group compared with the placebo group
By week 4
Thalidomide had induced a significant decrease in ESR and
decreased the number of children with
undernutrition when measured with weight- for-age z score less
than−1SD,but there were no other significant differences in the
secondary outcomes between treatment groups.

29. Efficacy Data

30. Efficacy Data

31. Independent predictors of treatment failure
Previous therapy with infliximab
PCDAI score >30
Weight-for-age z score <−1 SD
EIM

32. In the open label extension,of the 23 non responders to placebo, 2 needed surgery, whereas 21 given thalidomide
11 of 21 (52.4%) reached clinical remission at week 8 (P<.01).
Results of other outcomes comparing those crossed over to begin receiving thalidomide with the placebo group were similar to the outcomes of the randomized controlled trial

33. Longer-term Follow-up
Over all remission rate with thalidomide was 63.3% (31/49)
Overall, 32 children (65.3%) achieved 75% response
Mean time to reach remission was 10.1 weeks
Mean duration of clinical remission in thalidomide group was weeks v/s 6.3 weeks in the placebo group (P<.001)
No differences in the duration of remission in originally randomized to thalidomide and who began receiving it after failure of placebo (P = .90)
All the children had ceased receiving steroids by week 16
Thalidomide daily dose was progressively decreased during follow-up without losing clinical efficacy

34. Adverse Events
Nine severe adverse events requiring treatment suspension occurred, for a cumulative incidence of 2.1 per 1000 patient-weeks
Peripheral neuropathy was the most frequent severe adverse event.
Clinical neuropathy was observed with a minimum cumulative dose of 380 mg/kg (equivalent to 10 months of thalidomide therapy).
Most patients reached very high cumulative doses of thalidomide without developing clinical neuropathy

35. Other severe adverse events observed were amenorrhea, bradycardia, and 1 case of an acute neurologic event, interpreted as possible migraine or transient ischemic attack
Overall, non serious adverse events had a total cumulative incidence of 12.2 cases per 1000 patient-weeks

36. Adverse effects

37. Discussion
Children with refractory crohn’s disease account for about 30% of pediatric crohn’s disease cases and represent a subgroup with a higher risk of permanent impairment and higher health care costs for the individual and society
New effective and safe drugs are needed for these children.

38. Thalidomide was effective even in children with previous failure or intolerance to infliximab.
All these molecules have an antitumor necrosis factor α effect
Thalidomide also has an independent antiangiogenetic effect on vascular endothelial growth factor and basic fibroblastic growth factor, both of which are highly expressed in Crohn disease.

39. The poor response to placebo (11
The poor response to placebo (11.5%) confirms that sample represents cases of aggressive Crohn disease

40. Overall,this study suggests that safety of thalidomide in children with Crohn disease may be acceptable compared with that of other drugs. However, the study was clearly under powered to detect rare adverse events

41. Conclusions
Among children and adolescents with refractory Crohn disease, the use of thalidomide compared with placebo resulted in improved clinical remission at 8 weeks of treatment and longer term maintenance of remission in an open label follow- up.
These findings require replication to definitively determine the utility of this treatment

42. PCDAI Adult CDAI Abdominal pain Stools
Patient functioning, general well-being (Recall, 1 week)
Weight
Height
Abdomen
Peri-rectal disease
Hct (%)
ESR (mm/hr)
Albumin (g/L)
Adult CDAI
Abdominal pain
Stools
Patient functioning, general well-being (Recall, 1 week)
Weight
Abdomen mass
Extra-intestinal manifestations
Hct (%)
Antidiarrhoeals

43. Effect of magnesium supplementation and depletion on the onset and course of acute experimental pancreatitis
Schick V, et al. Gut 2013

44. Introduction
Acute pancreatitis, a fatal disease for 20% of severely affected patients
Premature and intracellular activation of digestive proteases induces tissue injury
Elevated concentrations of acinar cytosolic calcium are an important trigger of the disease
Ward JB, Lancet,1995

45. Under experimental conditions the removal of calcium from the incubation media or chelation of intracellular calcium prevents cellular injury
Mooren F,J Biol Chem 2003
On secretagogue stimulation, intracellular protease activation is primarily observed at the apical pole of acinar cells at the site where the stimulus-induced calcium release takes place
Kruger B, Am J Pathol 2000

46. Magnesium deficiency has been reported in individuals with chronic pancreatitis.
Papazachariou IM, Clin Chim Acta 2000
Increased intracellular magnesium concentrations directly influence the frequency and amplitude of calcium oscillations in response to cholecystokinin (CCK) or the acetylcholine analogue carbachol.
Mooren ,FASEB J, 2001

47. Aim
To investigate the effect of magnesium on premature enzyme activation in vitro as well as on pancreatic inflammation in vivo

48. MATERIALS AND METHODS

49. Cell isolation procedure
Pancreatic acini were prepared
After a 12-h fast animals were killed
Pancreas was rapidly removed
Pancreas minced into small pieces and placed into buffer

50. Measurment of intracellular calcium was done
Protease activity (intracellular trypsin and elastase activity) was measured in isolated acini
ATPase activity and kinase activity was measured in isolated acini

51. Animals and magnesium diets
Male Wistar rats were used
All rats were adjusted to laboratory conditions for 1 week prior to the experiments
Rats weighing 140–160 g were divided into four groups (n=15/ group)
Fed with diets containing the following concentrations of magnesium over 14 days:
1. magnesium low (<300 ppm)
2. magnesium normal (450 ppm)
3. magnesium high (1950 ppm)
4. magnesium very high ( ppm)

52. Male 14-week-old C56BL6/J mice were housed in metabolic cages for urine collection for Magnesium
Mice weighing 25–30 g were divided into four groups (n=10/group)
Pretreated over a period of 3 days with daily intraperitoneal MgSO4 at a concentration of –55.5, 192 or 384 mg/kg dissolved in 300 µL of NaCl 0.9%.
Control animals received 300 µL of NaCl 0.9% alone

53. Experimental pancreatitis
Rats after 14 days on the different magnesium diets were then randomly allocated in following groups:
1. infusion of saline (NaCl 0.9%) for 4 h
2. infusion of submaximal caerulein concentrations, 1
mcg/kg/h for 4 h
3. supramaximal stimulation with caerulein 10 mcg/kg/h
for 4 h
At the end of the infusion periods, the rats were killed
Blood samples were centrifuged at 4°C (3000g, 10 min), and serum was stored at −20°C
The entire pancreas was immediately removed ,trimmed of fat and divided into portions for the assays

54. Mice received up to eight hourly intraperitoneal injections of caerulein (50 mcg/kg) dissolved in 200 mcL NaCl 0.9% to induce a pancreatitis of greater severity
The mice were sacrificed
Blood samples were centrifuged at 4°C (3000g, 10 min) and serum was stored
The entire pancreas was immediately removed and divided into portions for assays.

55. Morphology
Tissuse sections (2 mcm) were stained with H&E and were evaluated by an observer unaware of the treatment groups.
Histomorphological evaluation of the specimens included the quantification of areas of necrosis

56. Evaluation of pancreatic oedema
Evaluated as pancreatic water content and was calculated by the wet-weight/dry-weight ratio
Initial weight (wet weight) was divided by the weight after desiccation at 160°C for 12 h (dry weight)

57. Ion measurements
Free serum calcium and magnesium concentrations were determined with an ion- specific electrode
Total magnesium content in pancreatic tissue was determined by atomic absorption spectroscopy (AAS)

58. Amylase and lipase activity
Measured photometrically with a Cobas Bio automated analyser

59. Trypsinogen activation peptide
TAP was quantified with the enzyme immunoassay

60. Tissue trypsin and elastase activity
For intercellular trypsin activity the substrate used was bis-(CBZ-Ile- Pro-Arg)-rhodamine110 (Invitrogen), and for elastase, bis-(CBZ-Ala4)-rhodamine110
The measurement of only intracellular protease activity was ensured by using phenylmethanesulfonylfluoride (PMSF) and aprotinin as inhibitors in the extracellular buffer

61. Myeloperoxidase activity
Myeloperoxidase(MPO) activity measurement was performed by spectrophotometry

62. Flow cytometric analysis
Intracellular staining was performed with the intracellular staining kit and anti-mouse/human FoxP3 APC
Cells were analyzed using FlowJo software
To determine serum concentrations of cytokines, a cytometric bead array was performed

63. Statistical analysis
All values in the figures represent means±SEM from at least four separate experiments
Differences between groups were compared using analysis of variance
Differences considered statistically significant with a p value <0.05

64. RESULTS
High extracellular magnesium levels (10 mM) resulted in a decrease in CCK-induced calcium transients, whereas low magnesium (0.2 mM) enhanced calcium transients and the frequency of calcium oscillations

66. Supramaximal doses of CCK resulted in the intracellular activation of trypsin and elastase and maximum activity was reached after about 30 min followed by a slow decrease in activity over the next 30 min.
The same experiment in the presence of 5 mM magnesium resulted in a significant decrease in intracellular trypsin and elastase activity

68. Those fed a low manegsium diet (<300 ppm magnesium) ,serum magnesium levels was 0.25±0.01 mM, whereas in those receiving the highest magnesium-containing diet ( ppm) we detected magnesium levels of 0.9±0.03 mM
Tissue magnesium levels increased in parallel but the increase did not reach significance

69. After induction of acute pancreatitis by intravenous infusion of supramaximal caerulein concentrations
Decrease in serum calcium level was most pronounced in animals fed the high magnesium chow

71. Supramaximal intravenous caerulein stimulation (10 mcg/kg/h) induced the characteristic increase in both serum amylase and serum lipase activity was pronounced in the magnesium-deficient rats
Amylase and lipase activities decreased with increasing dietary magnesium concentrations
Submaximal doses of caerulein (1 mg/kg bodyweight/h) were not sufficient to induce an increase in amylase and lipase levels in the animals on the high magnesium diet, whereas the same stimulus evoked a significant increase in both parameters in the animals on the low magnesium diet

73. Pancreatic oedema, which can be quantified by the wet/dry ratio
Control animals had a wet/dry ratio in the range of about 2.6–3.0
Submaximal stimulation with caerulein ,induced a significant increase of the wet/dry ratio only in the animals fed the low magnesium
Supramaximal stimulation with caerulein induced an increase in the wet/dry ratio in all animals, However, the increase was most prominent in the animals fed the low magnesium diet and decreased with increasing magnesium concentrations in the diet

74. Quantification of intracellular vacuole formation showed that after supramaximal caerulein stimulation the number of vacuoles increased most prominently in the magnesium-deficient group
However, even submaximal stimulation led to an increase in vacuole formation in the magnesium-deficient animals

78. Submaximal concentrations of caerulein effectively induced pancreatic TAP formation and urine TAP only in magnesium-deficient rats .
After supramaximal stimulation with caerulein,pancreatic TAP formation and raised urine TAP could be found in all animals, However, those on a high magnesium diet showed a significantly lower values.

80. Trypsin and Elastase activity was highest in the magnesium-deficient group
Enzymes activity also decreased in parallel with increasing magnesium content of the diet
Submaximal doses of caerulein were effective in inducing activation only in magnesium-deficient animals

82. To study the effect of magnesium in a more severe model of pancreatitis involving tissue necrosis, C57BL6/J mice were used with up to eight hourly supramaximal intraperito- neal caerulein injections (50 mg/kg bodyweight).
These mice were pretreated with three different concentrations of magnesium (55.5, 192 and 384 mg/kg bodyweight) intra peritoneally over a period of 72 h prior to the experiment

83. Results Increase in the concentration of urinary and serum Mg2+ levels
Reduced serum lipase levels at 1 h and 24 h of pancreatitis
Reduced areas of necrosis and less inflammatory infiltrates at 24 h after induction of pancreatitis
Expansion of FoxP3 positive T-cells which suggest an effect of magnesium on the immune system, possibly reducing a pro-inflammatory immune response

87. DISCUSSION
Mg predominantly interferes with calcium signalling and primarily acts on the cellular calcium influx in acinar cells, thereby controlling cellular enzyme secretion in pancreatic acinar cells
Present study demonstrated that magnesium not only regulates physiological mechanisms but also affects pathophysiological pathways.

88. Effect of magnesium in acinar cells is direct because calcium is replaced by magnesium in intracellular stores and predominantly involves calcium-dependent signalling events.
Magnesium can act as a natural calcium channel blocker that has a direct role in protecting mitochondria from calcium overload, improving their function and increasing the potential of ATP synthesis

89. Zhou et al demonstrated that submaximal doses of caerulein were effective in inducing acute pancreatitis in hypercalcaemic animals.
J Surg Res 1996
Papazachariou et al in chronic pancreatitis patients, which demonstrated that magnesium deficiency is more frequent in patients than in controls.
Clin Chim Acta 2000
Holtmeier reported three patients with chronic pancreatitis, hypomagnesaemia and abdominal pain
Schweiz Med Wochenschr,1968

90. Ryzen and Rude measured magnesium levels in serum and in peripheral blood mononuclear cells in 29 normocalcaemic or hypocalcaemic patients with acute pancreatitis and concluded that patients are frequently magnesium deficient in acute pancreatitis patients.

91. Conclusion
This study indicates that nutritional magnesium deficiency predisposes to acute pancreatitis, while high nutritional or parenteral magnesium can limit the morphological as well as the biochemical severity of pancreatitis
Magnesium could serve as a natural, inexpensive, orally bioavailable and well tolerated calcium antagonist, suitable for the treatment of pancreatitis, or even the prevention of pancreatitis in at-risk patients

92. EUOPAC2, clinical trials
Multi-centre randomised phase III, double blind, placebo controlled,
In patients diagnosed with hereditary  pancreatitis and idiopathic chronic pancreatitis
Hypothesis to be tested is a 30% reduction in the number of days due to pancreatitis from 12.5 days per year to less than nine days per year under the treatment with magnesium or an antioxidant cocktail called ANTOX.
A total of 240 patients will be randomised

93. MagPEP trial
Randomized, double-blind, placebo-controlled phase III study
To test whether the administration of intravenous magnesium sulphate before and after ERCP reduces the incidence and the severity of post-ERCP pancreatitis
Total 502 adult patients with ERCP will be randomized to receive either 4930 mg magnesium sulphate or placebo 60 min before and 6 hours after ERCP.
The incidence of clinical post-ERCP pancreatitis hyperlipasemia, pain levels, use of analgeics and length of hospital stay will be evaluated

94. PCDAI Adult CDAI Abdominal pain Stools
Patient functioning, general well-being (Recall, 1 week)
Weight
Height
Abdomen
Peri-rectal disease
Hct (%)
ESR (mm/hr)
Albumin (g/L)
Adult CDAI
Abdominal pain
Stools
Patient functioning, general well-being (Recall, 1 week)
Weight
Abdomen mass
Extra-intestinal manifestations
Hct (%)
Antidiarrhoeals