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

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 100000)

• 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

• 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

• 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

Aim

• To determine whether thalidomide is effective in inducing remission in refractory pediatric crohn’s 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

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

Inclusion Criteria

• Children and adolescents aged 2 to18 yrs

• Active crohn’s disease despite immunosuppresion

• Adverse events preventing continuous treatment

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

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

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

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

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

Adverse events

• Evaluation conducted at each visit A detailed history Vital signs Physical examination Laboratory analysis

• 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

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

Secondary End Points

• Mean PCDAI score• CRP level• ESR• BMI• Weight for age• Physician Global Assessment score• Incidence of adverse effects

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

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.

• 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

Flow chart of patient in trial

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

Baseline charcterstics

Baseline characterstics

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)

• At 4 weeks 25% and 75% responses were not different between groups• At week 8, Thalidomide group had a better response 25% response - 64.2% vs 30.8%75% response - 46.4% vs 11.5%( P <.01 for both)

PCDAI scores of groups by time

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.

Efficacy Data

Efficacy Data

Independent predictors of treatment failure

• Previous therapy with infliximab

• PCDAI score >30

• Weight-for-age z score <−1 SD

• EIM

• 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

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 181.1 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

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

• 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

Adverse effects

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.

• 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.

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

• 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

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

PCDAI• 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

Effect of magnesium supplementation and depletion on the onset and course of acute

experimental pancreatitis

Schick V, et al. Gut 2013

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

• 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

• 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

Aim

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

MATERIALS AND METHODS

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

• 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

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 (30 000 ppm)•

• 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

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

• 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.

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

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)

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)

Amylase and lipase activity

• Measured photometrically with a Cobas Bio automated analyser

Trypsinogen activation peptide

• TAP was quantified with the enzyme immunoassay

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

Myeloperoxidase activity

• Myeloperoxidase(MPO) activity measurement was performed by spectrophotometry

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

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

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

• 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

• 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 (30 000 ppm) we detected magnesium levels of 0.9±0.03 mM

• Tissue magnesium levels increased in parallel but the increase did not reach significance

• 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

• 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

• 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

• 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

• 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.

• 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

• 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

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

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.

• 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

• 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

• 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.

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

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

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

PCDAI• 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