Trastuzumab Duocarmazine in Pretreated Human Epidermal Growth Factor Receptor 2-Positive Advanced or Metastatic Breast Cancer: An Open-Label, Randomized, Phase III Trial (TULIP).

PURPOSE: Human epidermal growth factor receptor 2 (HER2)-targeted therapy is standard of care for HER2-positive (HER2+) breast cancer, but most patients develop progressive disease with persistent HER2 expression. No definitive treatment guidance currently exists beyond second line. Trastuzumab duocarmazine (T-Duo) is a third-generation, HER2-targeted antibody-drug conjugate that demonstrated efficacy and acceptable safety in phase I studies of heavily pretreated patients with HER2+/HER2-low breast cancer. METHODS: In this open-label, randomized, phase III trial, T-Duo was compared with physician's choice (PC) in patients with unresectable locally advanced/metastatic HER2+ breast cancer with progression during/after ≥2 HER2-targeted therapies or after trastuzumab emtansine (T-DM1). The primary endpoint was progression-free survival (PFS) by blinded independent central review. RESULTS: In total, 437 patients were randomly assigned 2:1 to T-Duo (n = 291) or PC (n = 146). The median age was 56.0 years (range, 24-86); most patients (93.6%) had metastatic disease. The median time from diagnosis of metastatic disease to trial entry was 3.5 years; the median number of prior HER2-targeted therapies in metastatic setting was three. The median PFS was 7.0 months (95% CI, 5.4 to 7.2) with T-Duo versus 4.9 months (95% CI, 4.0 to 5.5; hazard ratio [HR], 0.64 [95% CI, 0.49 to 0.84]; P = .002) with PC. PFS benefit was maintained across most predefined subgroups. The median overall survival (first analysis) was 20.4 (T-Duo) versus 16.3 months (PC; HR, 0.83 [95% CI, 0.62 to 1.09]; P = .153). Objective response rate was 27.8% (T-Duo) versus 29.5% (PC); other efficacy end points-clinical benefit rate, duration of response, and reduction in target lesion measurement-tended to favor T-Duo. Grade ≥3 treatment-emergent adverse events occurred in 52.8% (T-Duo) versus 48.2% (PC). CONCLUSION: Treatment with T-Duo was manageable, but tolerability was affected by prevalent ocular toxicity, leading to a higher discontinuation rate in the T-Duo arm. T-Duo significantly reduced the risk of progression in patients with advanced HER2+ breast cancer who have progressed during/after ≥2 HER2-targeted therapies or after T-DM1.

Switching from branded to generic glatiramer acetate: 15-month GATE trial extension results.

BACKGROUND: Open-label 15-month follow-up of the double-blind, placebo-controlled Glatiramer Acetate clinical Trial to assess Equivalence with Copaxone® (GATE) trial. OBJECTIVE: To evaluate efficacy, safety, and tolerability of prolonged generic glatiramer acetate (GTR) treatment and to evaluate efficacy, safety, and tolerability of switching from brand glatiramer acetate (GA) to GTR treatment. METHODS: A total of 729 patients received GTR 20 mg/mL daily. Safety was assessed at months 12, 15, 18, 21, and 24 and Expanded Disability Status Scale and magnetic resonance imaging (MRI) scans at months 12, 18, and 24. The presence of glatiramer anti-drug antibodies (ADAs) was tested at baseline and months 1, 3, 6, 9, 12, 18, and 24. RESULTS: The mean number of gadolinium-enhancing lesions in the GTR/GTR and GA/GTR groups was similar at months 12, 18, and 24. The change in other MRI parameters was also similar in the GTR/GTR and GA/GTR groups. The annualized relapse rate (ARR) did not differ between the GTR/GTR and GA/GTR groups, 0.21 and 0.24, respectively. The incidence, spectrum, and severity of reported adverse events did not differ between the GTR/GTR and GA/GTR groups. Glatiramer ADA titers were similar in the GTR/GTR and GA/GTR groups. CONCLUSION: Efficacy and safety of GTR is maintained over 2 years. Additionally, switching from GA to GTR is safe and well tolerated.

Equivalence of Generic Glatiramer Acetate in Multiple Sclerosis: A Randomized Clinical Trial.

IMPORTANCE: The patents for the first approved treatments for relapsing-remitting multiple sclerosis are expiring, creating the opportunity to develop generic alternatives. OBJECTIVE: To evaluate in the Glatiramer Acetate Clinical Trial to Assess Equivalence With Copaxone (GATE) study whether generic glatiramer acetate (hereafter generic drug) is equivalent to the originator brand glatiramer acetate (hereafter brand drug) product, as measured by imaging and clinical end points, safety, and tolerability. DESIGN, SETTING, AND PARTICIPANTS: Randomized, multicenter, double-blind, active and placebo-controlled phase 3 trial. The setting included academic medical centers and clinical practices. Participants were patients with relapsing-remitting multiple sclerosis 18 to 55 years old with at least 1 relapse in the prior year and 1 to 15 gadolinium-enhancing brain magnetic resonance imaging lesions. They were randomized between December 7, 2011, and March 21, 2013. The last participant completed follow-up December 2, 2013. INTERVENTIONS: Participants were randomized 4.3:4.3:1 to receive generic glatiramer acetate (20 mg), brand glatiramer acetate (20 mg), or placebo by daily subcutaneous injection for 9 months. MAIN OUTCOMES AND MEASURES: The primary end point was the total number of gadolinium-enhancing lesions during months 7, 8, and 9. Additional end points included other magnetic resonance imaging parameters, annualized relapse rate, and Expanded Disability Status Scale score. Safety and tolerability were assessed by monitoring adverse events, injection site reactions, and laboratory test results. RESULTS: In total, 794 participants were randomized and treated with generic drug (n = 353), brand drug (n = 357), or placebo (n = 84). The estimated mean numbers of gadolinium-enhancing lesions with generic drug and brand drug were lower than with placebo (ratio, 0.488; 95% CI, 0.365-0.651; P < 001), confirming study sensitivity. For gadolinium-enhancing lesions, the estimated ratio of generic drug to brand drug was 1.095 (95% CI, 0.883-1.360), which was within the predefined equivalence margin of 0.727 to 1.375. The incidence, spectrum, and severity of reported adverse events, including injection site reactions, were similar in the generic drug and brand drug groups. CONCLUSIONS AND RELEVANCE: As treatment for relapsing-remitting multiple sclerosis, glatiramer acetate generic drug and brand drug had equivalent efficacy, safety, and tolerability. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01489254.

Bilateral molecular changes in a neonatal rat model of unilateral hypoxic-ischemic brain damage.

Perinatal hypoxia ischemia (HI) is a frequent cause of neonatal brain injury. This study aimed at describing molecular changes during the first 48 h after exposure of the neonatal rat brain to HI. Twelve-day-old rats were subjected to unilateral carotid artery occlusion and 90 min of 8% O2, leading to neuronal damage in the ipsilateral hemisphere only. Phosphorylated-Akt levels were decreased from 0.5 to 6 h post-HI, whereas the level of phosphorylated extracellular signal-related kinases (ERK)1/2 increased during this time frame. Hypoxia-inducible factor (HIF)-1alpha protein increased with a peak at 3 h after HI. mRNA expression for IL-beta and tumor necrosis factor-alpha and -beta started to increase at 6 h with a peak at 24 h post-HI. Expression of heat shock protein 70 was increased from 12 h after HI onwards in the ipsilateral hemisphere only. Surprisingly, HI changed the expression of cytokines, HIF1-alpha ,and P-Akt to the same extent in both the ipsi- as well as the contralateral hemisphere, although neuronal damage was unilateral. Exposure of animals to hypoxia without carotid artery occlusion induced similar changes in cytokines, HIF-1alpha, and P-Akt. We conclude that during HI, hypoxia is sufficient to regulate multiple molecular mediators that may contribute, but are not sufficient, to induce long-term neuronal damage.

Selective inhibition of nuclear factor-kappaB activation after hypoxia/ischemia in neonatal rats is not neuroprotective.

Activated nuclear factor-kappaB (NFkappaB) has been shown to increase transcription of several genes that could potentially contribute to neuronal damage, such as proinflammatory cytokines, chemokines, and inducible nitric oxide synthase. The aim of our study was to investigate whether inhibition of NFkappaB activation could prevent hypoxia/ischemia (HI)-induced cerebral damage in neonatal rats. We used a cell permeable peptide (NEMO binding domain [NBD] peptide) that is known to prevent the association of the regulatory protein NEMO with IKK, the kinase that activates NFkappaB. Via this mechanism, the NBD peptide can specifically block the activation of NFkappaB, without inhibiting basal NFkappaB activity. Cerebral HI was induced in neonatal rats by occlusion of the right carotid artery followed by 90 min of hypoxia (Fio(2) = 0.08). Immediately upon reoxygenation, as well as 6 and 12 h later, rats were treated with vehicle or NBD peptide (20 mg/kg i.p.). Histologic analysis of brain damage was performed at 6 wk after HI. To assess NFkappaB activation, electromobility shift assays (EMSAs) were performed on brain nuclear extracts obtained 6 h after reoxygenation. Increased NFkappaB activity could be shown at 6 h after HI in both hemispheres. Peripheral administration of NBD peptide prevented this HI-induced increase in NFkappaB activity in both hemispheres. Histologic analysis of long-term cerebral damage revealed that inhibition of NFkappaB activation by administration of NBD peptide at 0, 6, and 12 h after HI resulted in an increment of neuronal damage. In conclusion, our data suggest that inhibition of NFkappaB activation using NBD peptide early after HI increases brain damage in neonatal rats.

Cerebral cortical tissue damage after hemorrhagic hypotension in near-term born lambs.

Hypotension reduces cerebral O(2) supply, which may result in brain cell damage and loss of brain cell function in the near-term neonate. The aim is to elucidate 1) to what extent the functional disturbance of the cerebral cortex, as measured with electrocortical brain activity (ECBA), is related to cerebral cortical tissue damage, as estimated by MAP2; and 2) whether there is a relationship between the glutamate, nitric oxide (NO), cGMP pathway and the development of cerebral cortical tissue damage after hemorrhagic hypotension. Seven lambs were delivered at 131 d of gestation. Hypotension was induced by withdrawal of blood until mean arterial blood pressure was reduced to 30% of normotension. Cerebral O(2) supply, consumption, and ECBA were calculated in normotensive conditions and after 2.5 h of hypotension. Concentrations of glutamate and aspartate in cerebrospinal fluid (CSF), NO(2) and NO(3) (NOx) in plasma, and cGMP in cortical brain tissue were determined in both conditions. CSF and brain tissue from siblings were used to determine normotensive values. Cortical neuronal damage was detected after 2.5 h of hypotension. ECBA was negatively related to the severity of the cortical damage. ECBA was related to respectively glutamate, NOx, and cGMP concentrations. In conclusion, cortical neuronal damage is detected after 2.5 h of hemorrhagic hypotension in the near-term born lamb. The damage is reflected by a reduction of ECBA. The glutamate, NOx, cGMP pathway is likely to be involved in the pathogenesis of cerebral cortical damage.

Expression of nitric oxide synthase isoforms and nitrotyrosine formation after hypoxia-ischemia in the neonatal rat brain.

BACKGROUND AND PURPOSE: Production of nitric oxide is thought to play an important role in neuroinflammation. Previously, we have shown that combined inhibition of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) can reduce hypoxia-ischemia-induced brain injury in 12-day-old rats. The aim of this study was to analyze changes in expression of nNOS, iNOS and endothelial NOS (eNOS), and nitrotyrosine (NT) formation in proteins in neonatal rats up to 48 h after cerebral hypoxia-ischemia. METHODS: Twelve-day-old rats were subjected to unilateral carotid artery occlusion and hypoxia, resulting in unilateral cerebral damage. NOS and nitrotyrosine expression were determined by immunohistochemistry and Western blot analysis at 30 min-48 h after hypoxia-ischemia. RESULTS: nNOS was increased in both hemispheres from 30 min to 3 h after hypoxia-ischemia. In the contralateral hemisphere, eNOS was decreased 1-3 h after hypoxia-ischemia. In the ipsilateral hemisphere, eNOS was decreased at 0.5 h after hypoxia-ischemia, normalized at 1-3 h and was increased 6-12 h after hypoxia-ischemia. At 24 and 48 h after hypoxia-ischemia, eNOS levels normalized. Surprisingly, iNOS expression did not change from 30 min up to 48 h after hypoxia-ischemia in the ipsi- or contralateral hemisphere. In addition, the regional expression of iNOS in the brain as determined by immunohistochemistry did not change after hypoxia-ischemia. Expression of nitrotyrosine was slightly increased in both hemispheres only at 30 min after hypoxia-ischemia. CONCLUSION: In 12-day-old rat pups, cerebral hypoxia-ischemia induced a transient increase in nNOS, eNOS, and nitrotyrosine in proteins, but no change in iNOS expression up to 48 h after the insult.

Long-term neuroprotection with 2-iminobiotin, an inhibitor of neuronal and inducible nitric oxide synthase, after cerebral hypoxia-ischemia in neonatal rats.

The short- and long-term neuroprotective effects of 2-iminobiotin, a selective inhibitor of neuronal and inducible nitric oxide synthase, were studied in 12-day-old rats following hypoxia-ischemia. Hypoxia-ischemia was induced by occlusion of the right carotid artery followed by 90 minutes of hypoxia (FiO2 0.08). Immediately on reoxygenation, 12 and 24 hours later the rats were treated with vehicle or 2-iminobiotin at a dose of 5.5, 10, 30, or 60 mg/kg per day. Histologic analysis of brain damage was performed at 6 weeks after hypoxia-ischemia. To assess early changes of cerebral tissue, levels of HSP70, nitrotyrosine, and cytochrome c were determined 24 hours after reoxygenation. Significant neuroprotection was obtained using a dose of 30 mg/kg per day of 2-iminobiotin. Levels of HSP70 were increased in the ipsilateral hemisphere in both groups (P<0.05), but the increase was significantly (P<0.05) less in the rats receiving the optimal dose of 2-iminobiotin (30 mg/kg per day). Hypoxia-ischemia did not lead to increased levels of nitrotyrosine, nor did 2-iminobiotin influence levels of nitrotyrosine. In contrast, hypoxia-ischemia induced an increase in cytochrome c level that was prevented by 2-iminobiotin. In conclusion, 2-iminobiotin administered after hypoxia-ischemia provides long-term neuroprotection. This neuroprotection is obtained by mechanisms other than a reduction of nitrotyrosine formation in proteins.

Hypoxia/ischemia modulates G protein-coupled receptor kinase 2 and beta-arrestin-1 levels in the neonatal rat brain.

BACKGROUND AND PURPOSE: Neurotransmitters, neuropeptides, chemokines, and many other molecules signal through G protein-coupled receptors (GPCRs). GPCR kinases (GRKs) and beta-arrestins play a crucial role in regulating the responsiveness of multiple GPCRs. Reduced expression of GRK and beta-arrestins leads to supersensitization of GPCRs and will thereby increase the response to neuropeptides and neurotransmitters. We analyzed GRK and beta-arrestin expression after cerebral hypoxia/ischemia (HI). MATERIALS AND METHODS: Twelve-day-old rat pups were exposed to 90 minutes of hypoxia (fraction of inspired oxygen [FiO2] 0.08) after ligation of the right carotid artery, a procedure that induces unilateral damage in the right hemisphere. At 6, 12, 24, and 48 hours after HI, the left (hypoxic) and right (hypoxic/ischemic) hemispheres were analyzed for GRK and beta-arrestin protein and mRNA expression by Western blotting and real-time polymerase chain reaction, respectively. In addition, we analyzed GRK2 expression in the hippocampus by immunohistochemistry. RESULTS: HI downregulated GRK2 protein expression in both hemispheres at 24 to 48 hours after HI, and the effect was more pronounced in the ipsilateral hemisphere. HI induced no global change in GRK6 protein expression. However, GRK2 was markedly decreased in the hippocampal region of the ipsilateral hemisphere that will be severely damaged after HI. No changes in global mRNA levels for GRK2 were detected. In contrast, HI increased beta-arrestin-1 protein expression as well as mRNA levels at 6 to 12 hours after HI. CONCLUSIONS: Neonatal HI-induced brain damage is associated with specific changes in the GPCR desensitization machinery. We hypothesize that these changes result in supersensitization of multiple GPCRs and might therefore contribute to HI-induced brain damage.

Redox state of near infrared spectroscopy-measured cytochrome aa(3) correlates with delayed cerebral energy failure following perinatal hypoxia-ischaemia in the newborn pig.

Early detection of delayed cerebral energy failure may be important in the prevention of reperfusion injury of the brain after severe perinatal hypoxia-ischaemia (HI). This study investigated whether monitoring of the redox state of cytochrome aa(3) (Cytaa(3)) with near infrared spectroscopy (NIRS) after severe perinatal asphyxia may allow us to detect early a compromised energy metabolism of the developing brain. We therefore correlated serial Cytaa(3) measurements (to estimate mitochondrial oxygenation) simultaneously with the (31)phosphorous-magnetic resonance spectroscopy ((31)P-MRS)-measured phosphocreatin/inorganic phosphate (PCr/Pi) ratio (to estimate cerebral energy reserve) in newborn piglets before and after severe hypoxia-ischaemia. The animals were treated upon reperfusion with either allopurinol, deferoxamine, or 2-iminobiotin or with a vehicle to reduce post-HI reperfusion injury of the brain. Four sham-operated piglets served as controls. Before HI, the individual Cytaa(3) values ranged between -0.02 and 0.71 micromol/L (mean value: -0.07) relative to baseline. The pattern over post-HI time of the vehicle-treated animals was remarkably different from the other groups in as far Cytaa(3) became more oxidised from 3 h after start of HI onwards (increase of Cytaa(3) as compared with baseline), whereas the other groups showed a significant reduction over time (decrease of Cytaa(3) as compared with baseline: allopurinol and deferoxamine) or hardly any change (2-iminobiotin and sham-operated piglets). Vehicle-treated piglets showed a significant reduction in PCr/Pi at 24 h after start of HI, but the cerebral energy state was preserved in 2-iminobiotin-, allopurinol- and deferoxamine-treated piglets. With severe reduction in PCr/Pi-ratio, major changes in the redox-state of Cytaa(3) also occurred: Cytaa(3) was mostly either in a reduced state (down to -6.45 micromol/L) or in an oxidised state (up to 6.84 micromol/L) at these low PCr/Pi ratios. The positive predictive value (PPV) of Cytaa(3) to predict severe reduction of the PCr/Pi ratio was 42%; the negative PPV was 87%. A similar relation was found for Cytaa(3) with histologically determined loss of neurons.

Pharmacological interventions in the newborn piglet in the first 24 h after hypoxia-ischemia. A hemodynamic and electrophysiological perspective.

The purpose of this study was to investigate whether combined inhibition of neuronal and inducible nitric oxide synthase (NOS) by 2-iminobiotin, free radical scavenging by allopurinol, and non-protein-bound iron chelation with deferoxamine improved cerebral oxygenation, electrocortical brain activity, and brain energy status during the first 24 h after hypoxia-ischemia (HI) in the newborn piglet. Forty-three newborn piglets were subjected to 1 h of severe HI by occluding both carotid arteries and phosphorous magnetic resonance spectroscopy ((31)P-MRS)-guided hypoxia, whereas five served as sham-operated controls. Upon reperfusion, piglets received vehicle (n=12), 2-iminobiotin (n=11), allopurinol (n=10), or deferoxamine (n=10). Cerebral oxygenation was recorded with near-infrared spectrophotometry (NIRS), electrocortical brain activity was assessed with amplitude-integrated EEG (aEEG), and cerebral energy status with (31)P-MRS. The oxygenated hemoglobin (HbO(2)) and total hemoglobin (tHb) were significantly increased in vehicle-treated piglets compared with 2-iminobiotin-treated and deferoxamine-treated piglets. No change in deoxygenated Hb (HHb) was demonstrated over time. The aEEG was significantly preserved in 2-iminobiotin- and deferoxamine-treated piglets compared with vehicle-treated piglets. Allopurinol treatment was not as effective as 2-iminobiotin treatment after HI. Phosphocreatine/inorganic phosphate ratios (PCr/P(i)) were significantly decreased for vehicle-treated piglets at 24 h post-HI, whereas 2-iminobiotin, allopurinol, and deferoxamine prevented the development of secondary energy failure. We speculate that the beneficial effects, especially of 2-iminobiotin, but also of deferoxamine, are due to reduced peroxynitrite-mediated oxidation.

Neuroprotection by selective nitric oxide synthase inhibition at 24 hours after perinatal hypoxia-ischemia.

BACKGROUND AND PURPOSE: Perinatal hypoxia-ischemia is a major cause of neonatal morbidity and mortality. Until now no established neuroprotective intervention after perinatal hypoxia-ischemia has been available. The delay in cell death after perinatal hypoxia-ischemia creates possibilities for therapeutic intervention after the initial insult. Excessive nitric oxide and reactive oxygen species generated on hypoxia-ischemia and reperfusion play a key role in the neurotoxic cascade. The present study examines the neuroprotective properties of neuronal and inducible but not endothelial nitric oxide synthase inhibition by 2-iminobiotin in a piglet model of perinatal hypoxia-ischemia. METHODS: Twenty-three newborn piglets were subjected to 60 minutes of hypoxia-ischemia, followed by 24 hours of reperfusion and reoxygenation. Five additional piglets served as sham-operated controls. On reperfusion, piglets were randomly treated with either vehicle (n=12) or 2-iminobiotin (n=11). At 24 hours after hypoxia-ischemia, the cerebral energy state, presence of vasogenic edema, amount of apparently normal neuronal cells, caspase-3 activity, amount of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)-positive cells, and degree of tyrosine nitration were assessed. RESULTS: A 90% improvement in cerebral energy state, 90% reduction in vasogenic edema, and 60% to 80% reduction in apoptosis-related neuronal cell death were demonstrated in 2-iminobiotin-treated piglets at 24 hours after hypoxia- ischemia. A significant reduction in tyrosine nitration in the cerebral cortex was observed in 2-iminobiotin-treated piglets, indicating decreased formation of reactive nitrogen species. CONCLUSIONS: Simultaneous and selective inhibition of neuronal and inducible nitric oxide synthase by 2-iminobiotin is a promising strategy for neuroprotection after perinatal hypoxia-ischemia.

Inhibition of nNOS and iNOS following hypoxia-ischaemia improves long-term outcome but does not influence the inflammatory response in the neonatal rat brain.

In this study, we tested the hypothesis that combined inhibition of nNOS and iNOS will reduce neuronal damage and the inflammatory response induced by perinatal hypoxia-ischaemia (HI). In 12-day-old rats, HI was induced by right carotid artery occlusion followed by 90 min of 8% O2. Immediately upon reoxygenation, the rats were treated with NOS inhibitors (n = 24) or placebo (n = 24). Neuropathology was scored at 6 weeks after HI on a 4-point scale (n = 12 per group). The expression of heat shock protein 70 (HSP70) and mRNA expression for cytokines were measured 12 h after HI (n = 12 per group). Histopathological analysis showed that the ipsilateral hemisphere in the NOS inhibition group was less damaged than in the placebo group (p < 0.05). HI induced a significant increase in HSP70 levels (p < 0.05) in the ipsilateral hemispheres, which tended to be lower in the NOS inhibition group (p = 0.07). HI induced an increase in mRNA expression for IL-1beta, TNF-alpha and TNF-beta, but there was no difference between the ipsi- and contralateral hemispheres. Combined inhibition of nNOS and iNOS did not induce any change in cytokine expression. We conclude that the long-term neuroprotective effects of combined nNOS and iNOS inhibition were not achieved by an altered cytokine response.

Effects of selective nitric oxide synthase inhibition on IGF-1, caspases and cytokines in a newborn piglet model of perinatal hypoxia-ischaemia.

Selective inhibition of neuronal and inducible nitric oxide synthase (NOS) with 2-iminobiotin previously showed a reduction in brain cell injury. In the present study, we investigated the effects of 2-iminobiotin treatment on insulin-like growth factor-1 (IGF-1) expression, caspase activity and cytokine expression in a newborn piglet model of perinatal hypoxia-ischaemia. Newborn piglets were subjected to 1 h of hypoxia-ischaemia and were treated intravenously with vehicle or 2-iminobiotin. Vehicle-treated piglets showed reduced IGF-1 mRNA expression and increased caspase-3 activity and DNA fragmentation. 2-Iminobiotin treatment, administered immediately upon reperfusion, prevented these observations. No differences in caspase-8 and -9 activity and cytokine [interleukin (IL)-1alpha/beta, IL-6, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta] mRNA expression were demonstrated between vehicle- and 2-iminobiotin-treated piglets at 24 h following hypoxia-ischaemia. IGF-1 mRNA correlated inversely with caspase-3 and transferase-mediated dUTP-biotin in situ nick end labelling score in the cortex, but positively with caspase-8. Cytokine mRNA did not correlate with IGF-1 mRNA, caspase-3 activity or DNA fragmentation. The present results indicate that the previously demonstrated neuroprotective effect of 2-iminobiotin treatment after perinatal hypoxia-ischaemia coincided with a preservation of the endogenous IGF-1 production and reduced caspase-3 activity, but not with a significant decrease in cytokine production.