Intracranial Hypertension and Cerebral Perfusion Pressure Insults in Adult Hypertensive Intraventricular Hemorrhage: Occurrence and Associations With Outcome.

OBJECTIVES: Elevated intracranial pressure and inadequate cerebral perfusion pressure may contribute to poor outcomes in hypertensive intraventricular hemorrhage. We characterized the occurrence of elevated intracranial pressure and low cerebral perfusion pressure in obstructive intraventricular hemorrhage requiring extraventricular drainage. DESIGN: Prospective observational cohort. SETTING: ICUs of 73 academic hospitals. PATIENTS: Four hundred ninety-nine patients enrolled in the CLEAR III trial, a multicenter, randomized study to determine if extraventricular drainage plus intraventricular alteplase improved outcome versus extraventricular drainage plus saline. INTERVENTIONS: Intracranial pressure and cerebral perfusion pressure were recorded every 4 hours, analyzed over a range of thresholds, as single readings or spans (≥ 2) of readings after adjustment for intracerebral hemorrhage severity. Impact on 30- and 180-days modified Rankin Scale scores was assessed, and receiver operating curves were analyzed to identify optimal thresholds. MEASUREMENTS AND MAIN RESULTS: Of 21,954 intracranial pressure readings, median interquartile range 12 mm Hg (8-16), 9.7% were greater than 20 mm Hg and 1.8% were greater than 30 mm Hg. Proportion of intracranial pressure readings from greater than 18 to greater than 30 mm Hg and combined intracranial pressure greater than 20 plus cerebral perfusion pressure less than 70 mm Hg were associated with day-30 mortality and partially mitigated by intraventricular alteplase. Proportion of cerebral perfusion pressure readings from less than 65 to less than 90 mm Hg and intracranial pressure greater than 20 mm Hg in spans were associated with both 30-day mortality and 180-day mortality. Proportion of cerebral perfusion pressure readings from less than 65 to less than 90 mm Hg and combined intracranial pressure greater than 20 plus cerebral perfusion pressure less than 60 mm Hg were associated with poor day-30 modified Rankin Scale, whereas cerebral perfusion pressure less than 65 and less than 75 mm Hg were associated with poor day-180 modified Rankin Scale. CONCLUSIONS: Elevated intracranial pressure and inadequate cerebral perfusion pressure are not infrequent during extraventricular drainage for severe intraventricular hemorrhage, and level and duration predict higher short-term mortality and long-term mortality. Burden of low cerebral perfusion pressure was also associated with poor short- and long-term outcomes and may be more significant than intracranial pressure. Adverse consequences of intracranial pressure-time burden and cerebral perfusion pressure-time burden should be tested prospectively as potential thresholds for therapeutic intervention.

A randomized 500-subject open-label phase 3 clinical trial of minimally invasive surgery plus alteplase in intracerebral hemorrhage evacuation (MISTIE III).

RATIONALE AND HYPOTHESIS: Surgical removal of spontaneous intracerebral hemorrhage may reduce secondary destruction of brain tissue. However, large surgical trials of craniotomy have not demonstrated definitive improvement in clinical outcomes. Minimally invasive surgery may limit surgical tissue injury, and recent evidence supports testing these approaches in large clinical trials. METHODS AND DESIGN: MISTIE III is an investigator-initiated multicenter, randomized, open-label phase 3 study investigating whether minimally invasive clot evacuation with thrombolysis improves functional outcomes at 365 days compared to conservative management. Patients with supratentorial intracerebral hemorrhage clot volume ≥ 30 mL, confirmed by imaging within 24 h ofknown symptom onset,and intact brainstem reflexes were screened with a stability computed tomography scan at least 6 h after diagnostic scan. Patients who met clinical and imaging criteria (no ongoing coagulopathy; no suspicion of aneurysm, arteriovenous malformation, or any other vascular anomaly; and stable hematoma size on consecutive scans) were randomized to either minimally invasive surgery plus thrombolysis or medical therapy. The sample size of 500 was based on findings of a phase 2 study. STUDY OUTCOMES: The primary outcome measure is dichotomized modified Rankin Scale 0-3 vs. 4-6 at 365 days adjusting for severity variables. Clinical secondary outcomes include dichotomized extended Glasgow Outcome Scale and all-cause mortality at 365 days; rate and extent of parenchymal blood clot removal; patient disposition at 365 days; efficacy at 180 days; type and intensity of ICU management; and quality of life measures. Safety was assessed at 30 days and throughout the study.

Efficacy and safety of minimally invasive surgery with thrombolysis in intracerebral haemorrhage evacuation (MISTIE III): a randomised, controlled, open-label, blinded endpoint phase 3 trial.

BACKGROUND: Acute stroke due to supratentorial intracerebral haemorrhage is associated with high morbidity and mortality. Open craniotomy haematoma evacuation has not been found to have any benefit in large randomised trials. We assessed whether minimally invasive catheter evacuation followed by thrombolysis (MISTIE), with the aim of decreasing clot size to 15 mL or less, would improve functional outcome in patients with intracerebral haemorrhage. METHODS: MISTIE III was an open-label, blinded endpoint, phase 3 trial done at 78 hospitals in the USA, Canada, Europe, Australia, and Asia. We enrolled patients aged 18 years or older with spontaneous, non-traumatic, supratentorial intracerebral haemorrhage of 30 mL or more. We used a computer-generated number sequence with a block size of four or six to centrally randomise patients to image-guided MISTIE treatment (1·0 mg alteplase every 8 h for up to nine doses) or standard medical care. Primary outcome was good functional outcome, defined as the proportion of patients who achieved a modified Rankin Scale (mRS) score of 0-3 at 365 days, adjusted for group differences in prespecified baseline covariates (stability intracerebral haemorrhage size, age, Glasgow Coma Scale, stability intraventricular haemorrhage size, and clot location). Analysis of the primary efficacy outcome was done in the modified intention-to-treat (mITT) population, which included all eligible, randomly assigned patients who were exposed to treatment. All randomly assigned patients were included in the safety analysis. This study is registered with ClinicalTrials.gov, number NCT01827046. FINDINGS: Between Dec 30, 2013, and Aug 15, 2017, 506 patients were randomly allocated: 255 (50%) to the MISTIE group and 251 (50%) to standard medical care. 499 patients (n=250 in the MISTIE group; n=249 in the standard medical care group) received treatment and were included in the mITT analysis set. The mITT primary adjusted efficacy analysis estimated that 45% of patients in the MISTIE group and 41% patients in the standard medical care group had achieved an mRS score of 0-3 at 365 days (adjusted risk difference 4% [95% CI -4 to 12]; p=0·33). Sensitivity analyses of 365-day mRS using generalised ordered logistic regression models adjusted for baseline variables showed that the estimated odds ratios comparing MISTIE with standard medical care for mRS scores higher than 5 versus 5 or less, higher than 4 versus 4 or less, higher than 3 versus 3 or less, and higher than 2 versus 2 or less were 0·60 (p=0·03), 0·84 (p=0·42), 0·87 (p=0·49), and 0·82 (p=0·44), respectively. At 7 days, two (1%) of 255 patients in the MISTIE group and ten (4%) of 251 patients in the standard medical care group had died (p=0·02) and at 30 days, 24 (9%) patients in the MISTIE group and 37 (15%) patients in the standard medical care group had died (p=0·07). The number of patients with symptomatic bleeding and brain bacterial infections was similar between the MISTIE and standard medical care groups (six [2%] of 255 patients vs three [1%] of 251 patients; p=0·33 for symptomatic bleeding; two [1%] of 255 patients vs 0 [0%] of 251 patients; p=0·16 for brain bacterial infections). At 30 days, 76 (30%) of 255 patients in the MISTIE group and 84 (33%) of 251 patients in the standard medical care group had one or more serious adverse event, and the difference in number of serious adverse events between the groups was statistically significant (p=0·012). INTERPRETATION: For moderate to large intracerebral haemorrhage, MISTIE did not improve the proportion of patients who achieved a good response 365 days after intracerebral haemorrhage. The procedure was safely adopted by our sample of surgeons. FUNDING: National Institute of Neurological Disorders and Stroke and Genentech.

African American Screening and Enrollment in (Clot Lysis: Evaluating Accelerated Resolution of Intraventricular Hemorrhage III) CLEAR III.

BACKGROUND: Under-enrolling minority patients in clinical trials reduces generalizability. CLEAR III, a randomized controlled trial, presented an opportunity to assess African American (AA) participation. METHODS: AA enrollment was compared to U.S. population and NINDS trial data then stratified by region; census data for 42 recruitment cities were compared to screening and randomization percentages, using simple linear regression. RESULTS: AAs were 25% of screens and 45.1% of enrollments (n=370), more than twice the 19.8% participation rate reported by the 2011 NINDS Advisory Panel on Health Disparities Research and triple the projected 13.9% 2014 U.S. population. Conversion rates were (AA vs. non-AA): overall (8.7% vs. 3.4%, p<0.001); Northeast (7.7% vs. 2.9%, p<0.001); South (8.2% vs. 4.0%, p<0.001); Midwest (10.3% vs. 3.6%, p<0.01); and West (8.9% vs. 3.8%, p=0.02). AA enrollments ranged from 0% to 100% (mean: 40.4%). AA screening ranged from 0% to 63.7% (mean: 23.2%). AA city census ranged from 1.3% to 82.7% (mean: 28.0%); higher census was associated with higher screening (p<0.0001) and enrollment (p=0.004). CONCLUSIONS: AAs were willing to enroll in an acute stroke trial. AA city census rates should be considered when selecting enrollment centers and setting recruitment goals. Factors leading to successful AA recruitment should be further investigated, as population-based participation is a goal in all trials.

Symptomatic Hemorrhagic Complications in Clot Lysis: Evaluation of Accelerated Resolution of Intraventricular Hemorrhage Phase III Clinical Trial (CLEAR III): A Posthoc Root-Cause Analysis.

BACKGROUND: As intraventricular thrombolysis for intraventricular hemorrhage (IVH) has developed over the last 2 decades, hemorrhagic complications have remained a concern despite general validation of its safety in controlled trials in the Clot Lysis: Evaluation of Accelerated Resolution of Intraventricular Hemorrhage Phase III (CLEAR-IVH) program. OBJECTIVE: To analyze factors associated with symptomatic bleeding following IVH with and without thrombolysis in conjunction with the recently completed CLEAR III trial. METHODS: We reviewed safety reports on symptomatic bleeding events reported during the first year after randomization among subjects enrolled in the CLEAR III trial. Clinical and imaging data were retrieved through the trial database as part of ongoing quality and safety monitoring. A posthoc root-cause analysis was performed to identify potential factors predisposing to rebleeding in each case. Cases were classified according to onset of rebleeding (during dosing, early after dosing and delayed), the pattern of bleeding, and treatment rendered (alteplase vs saline). RESULTS: Twenty subjects developed a secondary symptomatic intracranial hemorrhage constituting 4% of subjects. Symptomatic rebleeding events occurred during the dosing protocol (n = 9, 67% alteplase), early after the protocol (n = 5, 40% alteplase), and late (n = 6, 0% alteplase). Catheter-related hemorrhages were the most common (n = 7, 35%) followed by expansion or new intraventricular (n = 6, 30%) and intracerebral (n = 5, 25%) hemorrhages. Symptomatic hemorrhages during therapy resulted from a combination of treatment- and patient-related factors and were at most partially attributable to alteplase. Rebleeding after the dosing protocol primarily reflected patients' risk factors. CONCLUSION: Intraventricular thrombolysis marginally increases the overall risk of symptomatic hemorrhagic complications after IVH, and only during the treatment phase.

Demographic Risk Factors for Vascular Lesions as Etiology of Intraventricular Hemorrhage in Prospectively Screened Cases.

BACKGROUND: Spontaneous intraventricular hemorrhage (IVH) is associated with high rates of morbidity and mortality despite critical care and other advances. An important step in clinical management is to confirm/rule out an underlying vascular lesion, which influences further treatment, potential for further bleeding, and prognosis. Our aim is to compare demographic and clinical characteristics between IVH patients with and without an underlying vascular lesion, and among cohorts with different vascular lesions. METHODS: We analyzed prospectively collected data of IVH patients screened for eligibility as part of the Clot Lysis: Evaluation Accelerated Resolution of IVH Phase III (CLEAR III) clinical trial. The trial adopted a structured screening process to systematically exclude patients with an underlying vascular lesion as the etiology of IVH. We collected age, sex, ethnicity, and primary diagnosis on these cases and vascular lesions were categorized prospectively as aneurysm, vascular malformation (arteriovenous malformation, dural arteriovenous fistula, and cavernoma), Moyamoya disease, or other vascular lesion. We excluded cases <18 or >80 years of age. Baseline characteristics were compared between the CLEAR group (IVH screened without vascular lesion) and the group of IVH patients screened and excluded from CLEAR because of an identified vascular lesion. We further analyzed the differential demographic and clinical characteristics among subcohorts with different vascular lesions. RESULTS: A total of 10,538 consecutive IVH cases were prospectively screened for the trial between 2011 and 2015. Out of these, 496 cases (4.7%) screened negative for underlying vascular lesion, met the inclusion criteria, and were enrolled in the trial (no vascular etiology group); and 1,205 cases (11.4%) were concurrently screened and excluded from the trial because of a demonstrated underlying vascular lesion (vascular etiology group). Cases with vascular lesion were less likely to be >45 years of age (OR 0.28, 95% CI 0.20-0.40), African-American (OR 0.23, 95% CI 0.18-0.31), or male gender (OR 0.48, 95% CI 0.38-0.60), and more likely to present with primary IVH (OR 1.85, 95% CI 1.37-2.51) compared to those with no vascular etiology (p < 0.001). Other demographic factors were associated with specific vascular lesion etiologies. A combination of demographic features increases the association with the absence of vascular lesion, but not with absolute reliability (OR 0.1, 95% CI 0.06-0.17, p < 0.001). CONCLUSION: An underlying vascular lesion as etiology of IVH cannot be excluded solely by demographic parameters in any patient. Some form of vascular imaging is necessary in screening patients before contemplating interventions like intraventricular fibrinolysis, where safety may be impacted by the presence of vascular lesion.

Thrombolytic removal of intraventricular haemorrhage in treatment of severe stroke: results of the randomised, multicentre, multiregion, placebo-controlled CLEAR III trial.

BACKGROUND: Intraventricular haemorrhage is a subtype of intracerebral haemorrhage, with 50% mortality and serious disability for survivors. We aimed to test whether attempting to remove intraventricular haemorrhage with alteplase versus saline irrigation improved functional outcome. METHODS: In this randomised, double-blinded, placebo-controlled, multiregional trial (CLEAR III), participants with a routinely placed extraventricular drain, in the intensive care unit with stable, non-traumatic intracerebral haemorrhage volume less than 30 mL, intraventricular haemorrhage obstructing the 3rd or 4th ventricles, and no underlying pathology were adaptively randomly assigned (1:1), via a web-based system to receive up to 12 doses, 8 h apart of 1 mg of alteplase or 0·9% saline via the extraventricular drain. The treating physician, clinical research staff, and participants were masked to treatment assignment. CT scans were obtained every 24 h throughout dosing. The primary efficacy outcome was good functional outcome, defined as a modified Rankin Scale score (mRS) of 3 or less at 180 days per central adjudication by blinded evaluators. This study is registered with ClinicalTrials.gov, NCT00784134. FINDINGS: Between Sept 18, 2009, and Jan 13, 2015, 500 patients were randomised: 249 to the alteplase group and 251 to the saline group. 180-day follow-up data were available for analysis from 246 of 249 participants in the alteplase group and 245 of 251 participants in the placebo group. The primary efficacy outcome was similar in each group (good outcome in alteplase group 48% vs saline 45%; risk ratio [RR] 1·06 [95% CI 0·88-1·28; p=0·554]). A difference of 3·5% (RR 1·08 [95% CI 0·90-1·29], p=0·420) was found after adjustment for intraventricular haemorrhage size and thalamic intracerebral haemorrhage. At 180 days, the treatment group had lower case fatality (46 [18%] vs saline 73 [29%], hazard ratio 0·60 [95% CI 0·41-0·86], p=0·006), but a greater proportion with mRS 5 (42 [17%] vs 21 [9%]; RR 1·99 [95% CI 1·22-3·26], p=0·007). Ventriculitis (17 [7%] alteplase vs 31 [12%] saline; RR 0·55 [95% CI 0·31-0·97], p=0·048) and serious adverse events (114 [46%] alteplase vs 151 [60%] saline; RR 0·76 [95% CI 0·64-0·90], p=0·002) were less frequent with alteplase treatment. Symptomatic bleeding (six [2%] in the alteplase group vs five [2%] in the saline group; RR 1·21 [95% CI 0·37-3·91], p=0·771) was similar. INTERPRETATION: In patients with intraventricular haemorrhage and a routine extraventricular drain, irrigation with alteplase did not substantially improve functional outcomes at the mRS 3 cutoff compared with irrigation with saline. Protocol-based use of alteplase with extraventricular drain seems safe. Future investigation is needed to determine whether a greater frequency of complete intraventricular haemorrhage removal via alteplase produces gains in functional status. FUNDING: National Institute of Neurological Disorders and Stroke.

Participation of a coordinating center pharmacy in a multicenter international study.

PURPOSE: The activities of a coordinating center pharmacy (CCP) supporting a multicenter, international clinical trial are described. SUMMARY: Serving in a research support role comparable to that of a commercial clinical trial supply company, a CCP within the Johns Hopkins Hospital Investigational Drug Service (JHH IDS) uses its management expertise and infrastructure to support multicenter trials, such as the recently completed Clot Lysis: Evaluating Accelerated Resolution of Intraventricular Hemorrhage, Phase III (CLEAR III) trial. The role of the CCP staff in supporting the CLEAR III trial was overall investigational product (IP) management through coordination of IP-related operations to ensure high-quality care for study participants at study sites in the United States and abroad. For the CLEAR III trial, the CCP coordinated IP supply activities; provided education to site pharmacists; developed study-specific documents, including pharmacy manuals; communicated with trial stakeholders, including third-party IP distributors; monitored treatment assignments; and performed quality assurance monitoring to ensure compliance with institutional, state, federal, and international regulations regarding IP procurement and storage. Acting as a CCP for a multicenter international study poses a number of operational challenges while providing opportunities for the CCP to contribute to research of global importance and enrich the skill sets of its personnel. CONCLUSION: The development and implementation of the CCP at JHH IDS for the CLEAR III trial included several responsibilities, such as IP supply management, communication, and database, regulatory, and finance management.

Safety and efficacy of minimally invasive surgery plus alteplase in intracerebral haemorrhage evacuation (MISTIE): a randomised, controlled, open-label, phase 2 trial.

BACKGROUND: Craniotomy, according to the results from trials, does not improve functional outcome after intracerebral haemorrhage. Whether minimally invasive catheter evacuation followed by thrombolysis for clot removal is safe and can achieve a good functional outcome is not known. We investigated the safety and efficacy of alteplase, a recombinant tissue plasminogen activator, in combination with minimally invasive surgery (MIS) in patients with intracerebral haemorrhage. METHODS: MISTIE was an open-label, phase 2 trial that was done in 26 hospitals in the USA, Canada, the UK, and Germany. We used a computer-generated allocation sequence with a block size of four to centrally randomise patients aged 18-80 years with a non-traumatic (spontaneous) intracerebral haemorrhage of 20 mL or higher to standard medical care or image-guided MIS plus alteplase (0·3 mg or 1·0 mg every 8 h for up to nine doses) to remove clots using surgical aspiration followed by alteplase clot irrigation. Primary outcomes were all safety outcomes: 30 day mortality, 7 day procedure-related mortality, 72 h symptomatic bleeding, and 30 day brain infections. This trial is registered with ClinicalTrials.gov, number NCT00224770. FINDINGS: Between Feb 2, 2006, and April 8, 2013, 96 patients were randomly allocated and completed follow-up: 54 (56%) in the MIS plus alteplase group and 42 (44%) in the standard medical care group. The primary outcomes did not differ between the standard medical care and MIS plus alteplase groups: 30 day mortality (four [9·5%, 95% CI 2·7-22.6] vs eight [14·8%, 6·6-27·1], p=0·542), 7 day mortality (zero [0%, 0-8·4] vs one [1·9%, 0·1-9·9], p=0·562), symptomatic bleeding (one [2·4%, 0·1-12·6] vs five [9·3%, 3·1-20·3], p=0·226), and brain bacterial infections (one [2·4%, 0·1-12·6] vs zero [0%, 0-6·6], p=0·438). Asymptomatic haemorrhages were more common in the MIS plus alteplase group than in the standard medical care group (12 [22·2%; 95% CI 12·0-35·6] vs three [7·1%; 1·5-19·5]; p=0·051). INTERPRETATION: MIS plus alteplase seems to be safe in patients with intracerebral haemorrhage, but increased asymptomatic bleeding is a major cautionary finding. These results, if replicable, could lead to the addition of surgical management as a therapeutic strategy for intracerebral haemorrhage. FUNDING: National Institute of Neurological Disorders and Stroke, Genentech, and Codman.

A multicenter, randomized, double-blinded, placebo-controlled phase III study of Clot Lysis Evaluation of Accelerated Resolution of Intraventricular Hemorrhage (CLEAR III).

BACKGROUND: In adults, intraventricular thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) facilitates resolution of intraventricular haemorrhage (IVH), reduces intracranial pressure, decreases duration of cerebrospinal fluid diversion, and may ameliorate direct neural injury. We hypothesize that patients with small parenchymal haematoma volumes (<30 cc) and relatively large IVH causing acute obstructive hydrocephalus would have improved clinical outcomes when given injections of low-dose rtPA to accelerate lysis and evacuation of IVH compared with placebo. METHODS: The Clot Lysis Evaluation of Accelerated Resolution of Intraventricular Hemorrhage III trial is an investigator-initiated, phase III, randomized, multicenter, double-blind, placebo-controlled study comparing the use of external ventricular drainage (EVD) combined with intraventricular injection of rtPA to EVD plus intraventricular injection of normal saline (placebo) for the treatment of IVH. Patients with known symptom onset within 24 h of the computed tomography scan confirmed IVH and third or fourth ventricle obstruction, with or without supratentorial intracerebral haemorrhage volume <30 cc, who require EVD are screened with a computed tomography scan at least six hours after EVD placement and, if necessary, at consecutive 12-h intervals until stabilization of any intracranial bleeding has been established. Patients who meet clinical and imaging criteria (no ongoing coagulopathy and no suspicion of aneurysm, arteriovenous malformation, or any other vascular anomaly) will be randomized to either intraventricular rtPA or placebo. RESULTS: The primary outcome measure is dichotomized modified Rankin Scale 0-3 vs. 4-6 at 180 days. Clinical secondary outcomes include additional modified Rankin Scale dichotomizations at 180 days (0-4 vs. 5-6), ordinal modified Rankin Scale (0-6), mortality and safety events at 30 days, mortality at 180 days, functional status measures, type and intensity of intensive care unit management, rate and extent of ventricular blood clot removal, and quality of life measures.

Mechanism of an ATP-independent protein disaggregase: II. distinct molecular interactions drive multiple steps during aggregate disassembly.

The ability of molecular chaperones to overcome the misfolding and aggregation of proteins is essential for the maintenance of proper protein homeostasis in all cells. Thus far, the best studied disaggregase systems are the Clp/Hsp100 family of "ATPases associated with various cellular activities" (AAA(+)) ATPases, which use mechanical forces powered by ATP hydrolysis to remodel protein aggregates. An alternative system to disassemble large protein aggregates is provided by the 38-kDa subunit of the chloroplast signal recognition particle (cpSRP43), which uses binding energy with its substrate proteins to drive disaggregation. The mechanism of this novel chaperone remains unclear. Here, molecular genetics and structure-activity analyses show that the action of cpSRP43 can be dissected into two steps with distinct molecular requirements: (i) initial recognition, during which cpSRP43 binds specifically to a recognition motif displayed on the surface of the aggregate; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. This establishes a useful framework to understand the molecular mechanism by which binding interactions from a molecular chaperone can be used to overcome protein aggregates in the absence of external energy input from ATP.

Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin.

Understanding how the folding of proteins establishes their functional characteristics at the molecular level challenges both theorists and experimentalists. The simplest test beds for confronting this issue are provided by electron transfer proteins. The environment provided by the folded protein to the cofactor tunes the metal's electron transport capabilities as envisioned in the entatic hypothesis. To see how the entatic state is achieved one must study how the folding landscape affects and in turn is affected by the metal. Here, we develop a coarse-grained functional to explicitly model how the coordination of the metal (which results in a so-called entatic or rack-induced state) modifies the folding of the metallated Pseudomonas aeruginosa azurin. Our free-energy functional-based approach directly yields the proper nonlinear extra-thermodynamic free energy relationships for the kinetics of folding the wild type and several point-mutated variants of the metallated protein. The results agree quite well with corresponding laboratory experiments. Moreover, our modified free-energy functional provides a sufficient level of detail to explicitly model how the geometric entatic state of the metal modifies the dynamic folding nucleus of azurin.