Erythropoietin Therapy for Subarachnoid Hemorrhage
Erythropoietin Therapy for Subarachnoid Hemorrhage
ABSTRACT:

 Delayed ischemic deficits (DID) and strokes caused by low cerebral blood flow (CBF) are major
 sources of poor outcome following aneurysmal subarachnoid hemorrhage (SAH). DID are often
 accompanied by vasospasm and abnormalities in cerebrovascular autoregulation, an important
 reflex involved in the defense against low CBF. Assessment of vasospasm and impaired
 autoregulation can be conveniently measured non-invasively by use of transcranial Doppler
 (TCD) and the transient hyperaemic response test (THRT). Vasospasm and abnormalities in the
 THRT can predict those patients who are at risk of developing DID. In this study, the
 investigators wish to explore the neuroprotective and angiogenic effects of systemic
 erythropoietin (EPO) therapy on vasospasm and autoregulation following SAH, and examine
 whether any improvements translate into reduced incidences of DID and poor outcome. Eighty
 patients with SAH will be recruited over one year to receive three doses in the first week of
 either intravenous epoetin beta 30000 IU or placebo (0.9% saline) 50 ml/30 min as part of a
 randomized, double-blind, placebo-controlled trial. The investigators propose daily TCD
 assessment for detecting vasospasm and abnormal autoregulation. Outcome measures will examine
 the influence of EPO therapy on the incidence, severity, and duration of vasospasm, abnormal
 autoregulation, and DID.

 PURPOSE:

 This study is a randomized, double-blind, placebo-controlled clinical trial investigating the
 potentially beneficial effects of systemic recombinant human erythropoietin therapy (Epoetin
 beta, NeoRecormon®, Roche, 30000IU/50 ml/30 min, three times in the first week) on cerebral
 autoregulation and incidence of delayed ischemic deficits (DID) following aneurysmal
 subarachnoid haemorrhage (SAH).

 HYPOTHESIS Systemic recombinant human erythropoietin therapy can be used safely following SAH
 to ameliorate vasospasm, improve cerebral autoregulation, reduce DID, and facilitate
 neurological recovery.
BACKGROUND:

 1. Delayed ischaemia deficits in subarachnoid haemorrhage

 Seven thousand patients suffer SAH each year within the UK with young adults (<55 years)
 being equally affected. Cerebral vasospasm and related cerebral ischemia are the major
 causes of delayed morbidity and mortality in patients who survive the initial SAH
 episode. Previous studies have revealed that immediate low cerebral blood flow (CBF)
 accompanies SAH, particularly in those in coma. However, for better grade patients the
 low CBF is often delayed and associated with cerebral vasospasm [Knuckey 1985].
 Vasospasm often precedes the onset of DID which commonly occurs in the 2nd week after
 SAH [Knuckey 1985]. Autoregulation is the most accomplished reflex mechanism and
 reflects an intrinsic ability of the cerebral vessels to dilate in an attempt to
 maintain constant CBF in the face of poor cerebral perfusion. Following SAH, impairment
 of autoregulation affects prognosis [Lam 2000]. Indeed, daily assessment of
 autoregulation can help to identify patients at high risk of clinical deterioration
 [Smielewski 1997].

 Autoregulation can be conveniently measured non-invasively from the bedside using
 transcranial Doppler (TCD). Direct measures of middle cerebral artery flow velocities
 (FV) are predictive of DID; elevated mean FV (>120cms/sec) and increased ratio of mFV
 between MCA and extracranial internal carotid artery (ICA) (Lindegaard ratio >3) being
 associated with cerebral vasospasm and higher incidence of adverse events [Lindegaard
 1988]. The rapid increase of FV with time is also predictive, as is the relationship
 between FV and spontaneous changes in blood pressure (correlation index Mx) [Czosnyka
 2000].

 A more sophisticated measure of abnormal cerebrovascular haemodynamics involves the
 transient hyperaemic response test (THRT) [Giller 1991]. This is used routinely in our
 department where we have demonstrated a relationship between an abnormal THRT and
 clinical outcome following SAH [Lam 2000]. The THRT assesses the FV response to a manual
 carotid compression (Figure 1) and can be repeated within a few minutes providing a
 reliable and readily accessible method for assessing autoregulation. When THRT is used
 for day-to-day assessments for the same patient, this test can provide a valuable
 monitor of sequential changes in autoregulation.

 2. Autoregulation after SAH

 Autoregulation is often impaired after SAH, even in patients presenting in good clinical
 grade [Voldby 1985]. An abnormal THRT response commonly develops either early during
 days 0 to 3 (primary), or late around days 7 to 14 (secondary) after the initial bleed.
 Primary abnormalities in THRT are far more common in poor grade patients, while
 secondary abnormalities occur more regularly in the initially good grade patients who
 later develop signs of DID with clinical deterioration [Ratsep 2002]. Routine and daily
 assessment of autoregulation with TCD helps to identify patients at higher risk for DID.
 This delay in the evolution of an abnormal autoregulation response in better grade
 patients presents an opportunity to introduce early therapeutic strategies designed to
 offset the potentially harmful effects. Indeed, routine use of plasma expansion is
 already adopted with this aim in mind [Origitano 1990].

 3. Therapeutic opportunity; why recombinant human erythropoietin?

 Recently attention has been focused on potential therapeutic roles for endogenous brain
 proteins possessing neuroprotective properties. This neuroprotective approach of using
 erythropoietin (EPO) is aimed at protecting potentially viable brain tissue from apoptosis
 [Ehrenreich 2002]. EPO, a 34-kDa hydrophobic sialoglycoprotein, is responsible for the
 survival, proliferation, and differentiation of committed erythroid progenitor cells. It is
 produced by foetal liver and adult kidney, which is accelerated during hypoxia [Lacombe
 1999]. Because there is no pre-formed EPO storage, therefore, the control of EPO gene
 transcription involves complex interactions with hypoxia-inducible factor 1 (HIF-1), a
 transcription factor which is activated during hypoxia, hypoglycaemia, and seizures [Lacombe
 1999].

 EPO receptor (EPO-R), like other members of the haematopoietic receptor family, does not
 possess an endogenous tyrosine kinase activity. However, its close association with Jak2
 tyrosine kinase can induce a rapid tyrosine phosphorylation on a number of proteins [Lacombe
 1999]. Hypoxia can induce an increased EPO-R expression, which in turn causes increased
 sensitivity to EPO [Chin 2000].

 Both EPO and EPO-R are ubiquitously distributed in neural tissues [Digicaylioglu 1995].
 Except larger vessels, EPO-R is abundantly distributed in astrocytes, neurons, and cerebral
 capillary endothelia [Marti 1996, Yamaji 1996, Bernaudin 1999, Brines 2000].
 Immunohistochemistry shows that these distributions of EPO-R are consistent with the
 anatomical location of blood-brain barrier (BBB) [Brines 2000]. Such a condensed distribution
 of EPO-R within and surrounding the cerebral capillaries implies that a small amount of EPO
 is sufficient to mediate physiological function in the central nervous system [Chin 2000].
 The increased production of EPO in response to hypoxia and anemia by astrocytes and neurons
 corresponds to that which occurs in kidney and liver [Masuda 1994]. Thus, during hypoxia, EPO
 acts directly on cerebral capillary endothelial cells as an angiogenic factor through a
 paracrine mechanism from the astrocytes and an endocrine mechanism from the kidney
 [Digicaylioglu 1995].

 EPO protects nervous tissue under any conditions characterised by a relative deficiency of
 ATP [Brine 2000]. This neuroprotective effect is achieved by several mechanisms.

 1. EPO can induce expression of several glycolytic enzymes, which re-directs energy
 metabolism toward favouring survival during oxygen deprivation [Digicaylioglu 1995].

 2. During cerebral ischemia, the N-methyl-D-aspartate (NMDA) receptor-mediated glutamate
 toxicity is the major cause of neuron death [Morishita 1997]. Glutamate toxicity is in
 part mediated by nitric oxide (NO). EPO up-regulates the expression of antioxidant
 enzymes on a transcriptional level in vascular smooth muscle cells [Kusano 1999], and
 can potentially protect neurons from NO-generating agents [Sakanaka 1998]. Furthermore,
 EPO can suppress the increase in Ca2+ concentration induced by glutamate and the nitric
 oxide (NO)-induced apoptosis [Morishita 1997].

 EPO has been shown to provide neurotrophic [Konishi 1993] and angiogenic effects [Yamaji
 1996], and it has the potential for neural regeneration [Siren 2001]. These effects are
 dose-dependent and unrelated to the nerve growth factor (NGF) [Konoshi Y 1993]. The
 distributions of EPO and EPO-R correspond to the sequence of histopathological changes of
 cerebral ischemia/infarction. In acute hypoxia (cardiac arrest), strong immunoreactivity of
 EPO can be found in vascular endothelium, while the EPOR is expressed strongly in neurons
 [Siren 2001]. After cerebral ischemia, EPO plays an important role in angiogenesis and glial
 reaction. EPO immunoreactivity appears in the endothelium, intravascular inflammatory cells
 and neurons of the penumbra. Also EPO-R is expressed in neurons, astrocytes and endothelial
 cells [Bernaudin 1999, Siren 2001]. Because the expression of EPO-R precedes the
 up-regulation of EPO synthesis [Bernaudin 1999], the sensitivity to EPO increases [Chin
 2000]. In contrast, these changes are not detectable in non-ischemic cortex [Bernaudin 1999],
 indicating an increased potential for EPO to act where neurons are at risk [Siren 2001]. By
 protecting endothelial cells from apoptosis, EPO can increase cerebral blood flow, improve
 microcirculation of the penumbra, and improve tissue oxygenation by angiogenesis, thus
 significantly reduce the infarct volume [Bernaudin 1999, Siren 2001]. This angiogenesis
 corresponds to the finding that under hypoxic conditions such as exposure to high altitudes,
 the brain increases the mean microvascular density [Lamanna JC 1992]. In old infarcts, EPO
 and EPO-R are strongly expressed in reactive astrocytes. Thus the EPO system may participate
 in the repair process [Siren 2001]. The persistent up-regulation of EPO production by
 astrocytes after stroke also provides rapidly available sources of EPO to make neurons more
 tolerate ischemia [Siren 2001]. These endogenous neuroprotective mechanisms are insufficient
 to cope with acute injuries, as severe ischemic insult can exhaust the EPO/EPO-R
 neuroprotective capacity of the brain or the latency of de novo synthesis is too long.
 Clinical application of EPO should be administered rapidly to provide additional
 neuroprotection [Brines 2000].

 Epoetin beta is a 165-aa glycoprotein manufactured by using recombinant DNA technology, which
 contains the identical amino acid sequence of isolated human EPO and possesses the same
 biological activity. It is similar to the endogenous EPO except for minor differences in the
 pattern of 4 carbohydrate chains. [Brine 2000] The recombinant human EPO has been widely used
 for treating anaemia associated with chronic renal insufficiency, HIV infection, cancer, and
 surgery, and has an excellent safety record in wide clinical practice.

 4. Why 90,000 IU Epoetin beta?

 The safety and efficacy of epoetin beta for treating acute stroke (within 8 hours) in humans
 have been confirmed in a double blind, randomized, placebo-controlled clinical trial, which
 uses high dose intravenous epoetin beta (3,3000 IU/50 ml/30min/day, three days, with a
 cumulative dose 100,000IU) [Ehrenreich 2002]. No associated changes in blood pressure,
 haematocrit, haemoglobin, and red blood cell count are found in this trial [Ehrenreich 2002].
 Only the reticulocyte count increases 29.2 ±10.0% on the 4th day after the last dose of EPO
 [Ehrenreich 2002]. This systemic administration of EPO also has led to a 60- to 100-fold
 increase in CSF levels and does not require a breakdown of the blood-brain barrier (BBB)
 [Ehrenreich 2002]. Patients who are treated with EPO have a lower and earlier surge of serum
 S100 (secreted by reactive astrocytes and functional disturbances of membrane integrity of
 brain cell membrane) and thereafter return to within the normal limits sooner than the
 placebo [Ehrenreich 2002]. This earlier return of serum S100 towards the normal level implies
 that the inflammatory processes and/or BBB integrity can be improved more rapidly by systemic
 EPO therapy [Ehrenreich 2002]. The beneficial effects on stroke patients continue throughout
 30 days, particularly for those with moderate to severe strokes [Ehrenreich 2002].

 By activation of endothelial EPO receptors, the circulating EPO may appose inflammatory
 pathways in cerebral arteries and attenuate vasospasm induced by SAH. In vivo studies have
 found that systemic administration of EPO (i.p. 1000units/kg) immediately after SAH can
 improve survival and motor functions [Buemi 2000]. The systemic delivery of EPO has the
 advantage that it is universally available to the capillary endothelium in the whole brain,
 in contrast to intraventricular injection, which is highly localized and not practical in
 clinical settings [Brines 2000]. Because the concentration of EPO in CSF is independent of
 the serum level and the extent of BBB disruption in patients suffering from aneurysmal SAH,
 the EPO in CSF is predominantly produced by the brain in order to match its need [Springborg
 2003]. Therefore, an early activation of endothelial EPO-R may be a potential therapeutic
 strategy, which in fact has been proved by a reduction of S100 protein in CSF (a marker of
 brain damage) and restoration of cerebral autoregulation (for at least 48 hours) after
 systemic administration of recombinant human EPO [Springborg 2002]. Therefore, 90,000 Iuof
 epoetin beta will be use to achieve the maximal effects on CBF and autoregulation in these
 SAH patients to be studied.

 QUESTIONS TO BE ANSWERED:

 We wish to address the following hypotheses for SAH patients:

 1. Systemic treatment with epoetin beta (90,000IU) is safe following SAH

 2. Systemic treatment with epoetin beta reduces the incidence of abnormal FV and cerebral
 autoregulation following SAH.

 3. Systemic treatment with epoetin beta reduces the incidence of acute neurological
 deterioration following SAH If primary endpoints are improved with epoetin beta therapy,
 the data will be used to formulate the power calculation for design of a Phase III
 clinical outcome study. However, we will assess clinical outcome at the end of the trial
 and at discharge to detect any indication of adverse effect after EPO therapy.

 Randomisation procedure:

 Following informed consent, patients with angiography-positive aneurismal SAH will be
 randomised to receive either intravenous epoetin beta 30,000IU or placebo (0.9% saline)
 50ml/30min, three times in the first week after SAH (total dose 90,000IU). The number in each
 group will be 40. For blinding, the Pharmacy Manufacture Unit (PMU) will prepare and number
 identical vials containing either saline (0.9% NaCl) or epoetin beta reconstituted in saline.
 The vials will be randomly assigned to patients upon enrollment with the contents of each
 vial known only by the PMU. Trial medication will be started as soon as possible within 72
 hours of the ictus. As approximately 70% of aneurysms will be treated with open clipping, and
 the remainder with endovascular coils, we do not consider the method of treatment to
 represent a contaminating factor, but it will be included in the final analysis. Location,
 size, and morphology of the culprit aneurysm are not believed to affect outcome in our
 institution.

 Following randomisation and start of trial therapy the clinical management of each patient
 will be as routine. Arterial blood pressure will be continuously monitored (Finapress, or via
 radial arterial line).

 Safety:

 The full blood cell count, reticulocyte count, blood viscosity, coagulation profile, serum
 biochemistry, serum iron levels, and C-reactive protein (CRP) at the time of admission will
 be checked as clinical routine. Although EPO has effects of erythropoiesis and
 thrombopoiesis, associated deterioration or adverse events have not been observed in
 short-term treatment [Ehrenreich 2002]. However, in the face of any abnormalities the trial
 drug will be stopped and the safety committee informed. A safety committee (chaired by Dr Ken
 Smith, Consultant nephrologist) will review the safety data at monthly intervals or, if
 concerns arise, on a patient-by-patient basis.

 Primary outcome measures: Vasospasm and abnormalities in AR:

 Trial patients will be examined daily with TCD (DWL, Germany) using a 2-MHz probe mounted on
 a purposed head frame for two weeks since SAH ictus. The systolic, diastolic, and mean FV
 will be recorded (trans-temporal) by a single user (MT). Vasospasm will be defined as mean FV
 > 120 cm/sec and Lindegaard ratio >3 [Lindegaard 1988]. The regression index (Mx) between
 mean FV and spontaneous changes in ABP will be calculated. Two carotid compressions lasting 5
 seconds will be performed. The criteria for an acceptable THRT includes a sudden decrease in
 middle cerebral artery FV at the onset of compression, a stable TCD signal during
 compression, and a minimum of 30% decrease in FV with no blood pressure instability
 [Smielewski 1997]. The THRT ratio (THRR) is calculated using the formula: THRR = FVs
 (hyperaemia) / FVs(baseline), where FVs denotes systolic FV (Figure 1). THRR is classified as
 normal (≥l.10) or impaired (<1.10), and will be repeated 2 minutes later. The average value
 of the two tests will be recorded. Quality issues concerning the THRT response have been
 extensively evaluated in this laboratory [Smielewski 1997].

 Secondary endpoint measures: Development of DID:

 The clinical progress of each patient will be monitored daily. The development of a focal
 neurological deficit and/or a drop in the Glasgow Coma Scale by 2 points or more following
 vasospasm will be the criteria adopted to define an episode of DID [Pickard 1989]. Other
 factors which can affect consciousness, including systemic infection and infection of central
 nervous system, will be recorded for final analysis. Clinical outcomes will be assessed at
 the end of the trial and at the time of discharge. Durations of hospitalisation and NCCU stay
 will be observed.

 Statistical Analysis:

 The analysis will be performed by using statistical software, STATA (Texas, USA). Data will
 be expressed as mean±standard deviation. The multivariate analysis will be performed on the
 timings of vasospasm and abnormal AR, which will be computed as the number of days between
 the date of diagnosis of SAH and the date of the first detection of vasospasm and abnormal
 autoregulation in respect of variables likely to influence their occurrence: age, WFNS grade,
 Fisher's grade on CT scans, and treatment procedures, intraventricular haemorrhage and/or
 hydrocephalus by using the Cox Proportional Hazards regression. The measure of outcome will
 be the hazards ratio (HR), which expresses the probability of vasospasm or abnormal
 autoregulation for a specific category relative to the placebo group. Variables associated
 with EPO treatment (haematocrits, erythrocyte counts, reticulocyte counts, thrombocyte
 counts, serum iron levels, and viscosity) will be collected. The improvement in fit due to
 each variable will be tested for statistical significance at the 5% level with the likelihood
 ratio test. The test for departure from a linear trend (one degree of freedom) was used to
 assess the potential linear trend in certain categorical variables. Daily results from each
 patient will also be averaged to produce a patient-oriented database to satisfy the
 independence assumption for linear regression. The day-to-day variations in parameters will
 be calculated as the standard deviation divided by the average value from all examinations in
 each patient.

 Power analysis:

 Our previous observations indicate that vasospasm and abnormal THRT occur in 64% and 90% SAH
 of patients respectively. To demonstrate a 50% reduction in duration of vasospasm or abnormal
 autoregulation with a power of 80% and a significance level of 5% we will require
 examinations from 80 patients. This number of patients also has an 83% chance of
 demonstrating a 50% reduction in the incidence of clinical DID in the first 2 weeks following
 the ictus. The study is not powered to show a reduction in clinical endpoints although trends
 will be sought to identify any potential adverse effects.

 DETAILS OF ANY DIFFICULTIES THAT CAN BE FORESEEN

 1. Patient recruitment:

 The Neurosurgical Unit at Addenbrookes treats 80 aneurysmal SAH patients per annum.
 Previous studies in our unit have shown high ascertainment, and we were the largest
 contributor to the International Hypothermia for Aneurysm Surgery Trial (IHAST). We do
 not foresee difficulties with recruitment.

 2. Safety issues:

 The safety and efficacy of using high dose intravenous epoetin beta (100,000IU) for
 treating human acute stroke (within 8 hours) have been confirmed [Ehrenreich 2002]. The
 therapeutic margin of EPO is very wide. Even at very high serum levels no adverse
 effects have been observed [NeoRecormon information sheet, Roche].

 3. Assessment of AR using TCD:

 Quality control for THRT assessment has been vigorously addressed, failure to complete the
 test is rare [Ratsep 2002]. The incidence of significant carotid atherosclerotic disease
 precluding safe carotid compression is less than 1% [Lam 2000]. The test is well tolerated
 and we know of no patient who requested to withdraw from the investigation in previous
 studies due to discomfort etc.

 FUTURE RESEARCH

 Results from this phase II study will be used to design a phase III clinical outcome trial
 using the Spontaneous Intracranial Haemorrhage Group mechanism. Any potential benefits may
 extrapolate to other cerebrovascular conditions where low CBF states and loss of
 autoregulation occur (e.g. acute cerebral vasculitis).
Inclusion Criteria:

 -  Adult patients (>= 18 years)

 -  Aneurysmal subarachnoid hemorrhage

 Exclusion Criteria:

 -  Uncontrolled systemic hypertension (systolic blood pressure > 220 mmHg)

 -  Erythrocytosis vera

 -  Concurrent erythropoietin therapy

 -  Negative angiography

 -  Subarachnoid hemorrhage more than 7 days