Brain Interstitium Temozolomide Concentration Pre and Post Regadenoson Administration

Blood brain barrier:

The blood-brain barrier (BBB) is an intricate barricade composed of three distinct cell
types: brain endothelial cells, pericytes, and astrocyte foot processes. While molecules that
are small and lipophilic may easily traverse the BBB, large (> 180 Daltons) and/or
hydrophilic particles require active transport, or receptor mediation. Specifically, the BBB
integrity and degree of permeability is regulated by the capillary endothelial cells in
response to astrocytic signals. The strength of intercellular junctions (ex. tight junctions,
adherins) amongst endothelial cells also plays a major role in permeability. Therefore,
modulation of all these paracellular properties may serve a large part in decreasing BBB
integrity and thus improving drug penetration to the tumor bed.

Rationale for Regadenoson:

Adenosine may be a plausible means to transiently disrupt the BBB. Adenosine function is
regulated by its four structurally related G-protein coupled receptors: A1, A2A, A2B and A3.
Specifically, A1 and A2A have high expression levels within the brain. In 2011, Carman et al
demonstrated, with the selective A2A receptor agonist, Regadenoson, increased BBB
permeability to 70kD Dextran in both mice and rat brains. The large molecule of Dextran was
seen in the brain for up to 180 minutes following a single injection in both mice and rats.
Studies performed at Johns Hopkins have also demonstrated a transient disruption of the BBB
from Regadenoson in rodents by measuring temozolomide concentrations. The investigators found
that brain/plasma concentrations in rats post Regadenoson resulted in a 1.6 fold increase of
temozolomide compared to temozolomide given alone in normal rat brains. Future animal studies
will evaluate varied chemotherapy agents within the brain and brain tumor with and without
Regadenoson.

Currently, Regadenoson is approved by the US Food and Drug Administration as a coronary
vasodilator, for myocardial perfusion imaging in patients with suspected myocardial coronary
artery disease. Single-photon emission computed tomography (SPECT) imaging performed with
99mTc-sestamibi radiotracer is used to measure coronary blood flow both at rest and stress
conditions with Regadenoson. While the adverse event profile is similar to adenosine, the
most common side effects of Regadenoson are flushing, chest pain, dyspnea and headaches.

Intracerebral Microdialysis:

Plasma drug concentration measurements are an unreliable method to assess delivery of drugs
across the BBB. In contrast, intracerebral microdialysis monitoring allows for approximate
measurements within extracellular fluid (ECF) sampling of the brain. Microdialysis placement
within the brain is not a novel technique and has been utilized routinely in the intensive
care unit setting to measure brain metabolism by sampling of ECF of traumatic brain injury
patients. Microdialysis catheters are now FDA approved and are being placed routinely with
intracranial pressure monitors. This method allows for continuous measurement of ECF within a
tumor or normal tissue. The dialysis probe has a semipermeable membrane which is less than 1
mm in diameter into which two sections of microcatheter are fused. Previous studies have
demonstrated the feasibility of keeping the catheters in place of critically injured patients
for up to 2 weeks. When placed at the time of surgical resection, the microcatheters are
sterotactically implanted, placing the probe within the desired brain and/or tumor region.
Externally, the catheter is connected to a syringe pump, which delivers a low flow rate
(µl/min) of continuous perfusion fluid (Lactated Ringers or artificial CSF) and dialysate is
collected in a microvial from the outlet tube. This sterile, single use catheter is minimally
invasive and developed to achieve optimal diffusing characteristics similar to passive
diffusion of a capillary blood vessel. Just as in the function of brain capillary vessel,
water, inorganic ions and small organic molecules freely diffuse across the membrane of the
probe, whereas proteins and protein bound compounds are impermeable. Additionally, lipophilic
compounds are poorly recovered.

Use of Intracerebral Microdialysis to Quantitate temozolomide Concentrations Temozolomide is
a FDA approved oral alkylating agent used in upfront and relapsed therapy of adult and
pediatric patients with high grade glioma. While temozolomide (TMZ) with radiotherapy has
assisted with improving overall survival rates of high grade gliomas, previous studies have
proven approximately only 20% of the drug crosses the BBB after peripheral administration.
The peak concentration of TMZ in the brain has been shown to be approximately 1-2 hours after
ingestion. Once ingested, TMZ undergoes degradation from its prodrug form to the highly
reactive alkylating agent, methyl-triazenyl imidazole carboxamide (MTIC). Previous studies
have utilized brain microdialysis catheters to measure TMZ brain interstitium levels post
primary or metastatic brain resection/biopsy. Both serum and dialysates were collected at 30
minute time intervals and analyzed to quantitate TMZ drug delivery to the tumor bed and
surrounding tissue yet due to drug stability issues, MTIC concentrations were not feasible
for measurement in the microdialysis setting. Thus, the investigators propose to measure TMZ
concentrations pre and post Regadenoson administration with the hypothesis that transient BBB
disruption will result in improved drug efficacy to the brain interstitium and tumor bed. If
gross total resection cannot be achieved two microdialysis catheters will be placed after
resection. One microdialysis catheter will be placed in the MRI enhancing tumor area and one
catheter will be placed in a non-enhancing area, allowing for later assessment of the degree
of BBB permeability both in the tumor and normal brain. These studies will be beneficial to
evaluate the role of increasing temozolomide delivery in an effort to further increase tumor
cytotoxicity and progression-free survival in such an aggressive tumor.

Use of Intracerebral Microdialysis to Quantitate Markers of Neuroinflammation Previous
studies have demonstrated that patients with increased inflammatory cytokines from medical
illnesses exhibit increased rates of depression. Both chronic and acutely elevated levels of
inflammatory cytokines can result in the development of neuropsychiatric dysfunction,
including cognitive slowing, inattention, or depression. Specifically, Dowlati et al
concluded from a meta-analytic study that a small proportion of patients with idiopathic
major depressive disorder exhibited increased serum cytokine levels. Thus, the investigators
propose to quantitate markers of neuroinflammation and oxidative stress in collected
dialysate, with the hypothesis that variance in these levels may correlate with affective
and/or cognitive dysfunction.

Risks of Microdialysis:

These studies will be a novel investigation in the use of brain microdialysis to measure TMZ
concentration in the post-operative setting with combinational administration of Regadenoson
and temozolomide. To minimize risks post-surgical resection or biopsy, patients will only
receive temozolomide 150mg/m2 twice, which is less than half the standard 5 day course
administered during maintenance therapy for malignant glioma post radiation therapy. In
addition, therapy will be given for only two days in the ICU setting, with minimal tumor
toxicity anticipated with a suboptimal treatment dose over a short duration.[ The procedures
involved with the insertion and removal of the dialysis probe can be performed with minimal
injury at the time of surgery.[18,19] Although insertion of the microdialysis probe could
conceivably disrupt the BBB, this has not proven to be problematic, as demonstrated by rapid
normalization of the BBB after placement, allowing for equilibration and minimization of
artifact.[20-22] Although it can be safely performed, microdialysis is nevertheless an
invasive surgical procedure with a small risk of bleeding at the site of insertion and
discomfort for the patient. The previous study which evaluated brain interstitium
concentrations of TMZ evaluated dialysate samples from seven patients. All of the patients
tolerated placement of the catheters and collection of the dialysate samples well, yet 1
patient developed several grade 3 and 4 neurologic adverse events whose etiologies remain
unclear. All of the neurologic deficits improved without completely resolving.

Inclusion Criteria:

- Age ≥ 18

- Diagnosis of recurrent high grade glioma confirmed by frozen pathology
intra-operatively

- Patients with planned surgical debulking or biopsy

- Karnofsky performance status (KPS) ≥ 60%

- Mini Mental Status Exam ≥ 15

- Adequate hepatic function

- Total bilirubin ≤ 1.8 mg/dL

- Serum levels of aspartate aminotransferase ≤ 3x the institutional upper limit of
normal

- Adequate renal function

- Serum creatinine ≤ 1.5mg/dL

- Adequate bone marrow function

- ANC ≥ 1500 cells/mm3

- Platelet count ≥ 100,000 cells/mm3

Exclusion Criteria:

- Currently receiving chemotherapy, or radiation therapy

- Allergic to temozolomide

- Pregnant or breast-feeding

- Have a serious medical or psychiatric illness or social situation that could interfere
with catheter placement or monitoring

- Prior use of VEGF or VEGFR-targeted therapy

- Use of medications: Vincristine, Vinblastine, Rifampin, Opioid's, Antidepressants
within the past 24 hours

- Use of investigational agents within the past 4 weeks

- NCI CTC Grade 3 or greater baseline neurologic symptoms

- History of cardiac disease:

- Atrial fibrillation or uncontrolled tachyarrhythmia, or advanced atrioventricular
block (second or third degree heart block)

- History of sinus node dysfunction

- History of symptomatic sinus bradycardia or recorded (heart rate <40/minute)

- Use of dipyridamole, dipyridamole-containing medications

- Any history of coronary heart disease defined by prior myocardial infarction or
ischemia on cardiac stress testing or coronary stenosis ≥50% severity

- Moderate or severe aortic stenosis

- Uncontrolled hypertension (BP >200/110)

- Decompensated or inadequately controlled congestive heart failure.

- Acute pulmonary embolism

- Acute myocarditis or pericarditis

- Acute aortic dissection

- Severe pulmonary hypertension

- Hypertrophic obstructive cardiomyopathy

- Electrolyte abnormalities

- Unexplained chest pain that may be anginal in nature

- Use of sublingual nitroglycerin

- History of Regadenoson hypersensitivity

- Known symptomatic hypotension, uncontrolled hypertension within 30 days

- History of moderate to severe bronchospastic lung disease (including moderate to
severe asthma)

- Use of aminophylline in the last 24 hours

- Use methylxanthine or caffeine in the last 12 hours

- Contraindication to adenosine (e.g., bronchoconstrictive/bronchospastic disease)

- Use of potassium supplements or potassium sparing medications in the past 24 hours

- Any patient not deemed medically stable by a physician

- Recent history of illicit drug use (past 3 months)