Patent Application
20080286202
The present invention relates generally to the field of diagnosis in a patient exhibiting signs of clinical dementia. In particular, the application is directed to a method for imaging areas of amyloid deposition in patients exhibiting clinical signs of dementia in pre-diagnosed states, such as mild cognitive impairment (MC1), or in a dementing disorder of questionable etiology and comparing the data obtained with normative levels in a control subject.
A. Diseases Associated with Amyloid Deposition
A condition closely related to Alzheimer's Disease (AD) is characterized by either isolated memory impairment or impairment in several cognitive domains, but not of sufficient severity to meet diagnostic criteria for Alzheimer's disease. This condition has been termed mild cognitive impairment and may represent a prodromal phase of AD. Mild cognitive impairment is defined as an intermediate or transitional state from a normal cognitive state to dementia. Subjects with a mild cognitive impairment (MC1) typically have a memory impairment beyond that expected for age and education yet are not demented.
There is some indication that patients diagnosed as mild cognitive impairment will progress to AD. There is also indications that mild cognitive impairment may represent a complex heterogeneous condition and that some patients with mild cognitive impairment will not develop AD or other dementing disorders.
There have been volumes of interest in discerning the boundary of dementia to AD. Most of the interest deals with a boundary or transitional state between normal aging and dementia, or more specifically, Alzheimer disease (AD). Reviews of several studies have indicated that these individuals are at an increased risk for developing AD ranging from 1% to 25% per year. The variability in these rates likely reflects differing diagnostic criteria, measurement instruments, and small sample sizes. See Dawe et al., Int'l J. Geriatr. Psychiatry 7: 473 (1992).
Patients diagnosed with an MCI are also becoming of interest for treatment trials. The Alzheimer's Disease Cooperative Study, which is a National Institute on Aging consortium of Alzheimer's Disease research groups, is embarking on a multicenter trial of agents intended to alter the progression of patients with MCI to AD. See Grundman et al., Neurology, 1996, A403.
Questions can be raised as to the diagnostic criteria for MC1. Some investigators believe that virtually all these patients with mild disease have AD neuropathologically, and, therefore, this may not be a useful distinction. See Morris et al., Neurology 41: 469 (1991). Others note that, while many of these patients progress to AD, not all do and, consequently, that the distinction is an important one. See Grundman, ibid; Petersen et al., JAMA 273: 1274 (1995); Petersen et al., Ann NY Acad. Sci. 802: 58 (1996).
AD is believed to afflict some 4 million Americans and perhaps 20-30 million people worldwide. AD is recognized as a major public health problem in developed nations.
AD is a neurodegenerative illness characterized by memory loss and other cognitive deficits. McKhann et al., Neurology 34: 939 (1984). It is the most common cause of dementia in the United States. AD can strike persons as young as 40-50 years of age, yet, because the presence of the disease is difficult to determine without dangerous brain biopsy, the time of onset is unknown. The prevalence of AD increases with age, with estimates of the affected population reaching as high as 40-50% by ages 85-90. Evans et al., JAMA 262: 2551 (1989); Katzman, Neurology 43: 13 (1993).
Neuropathologically, AD is characterized by the presence of neuritic plaques (NP), neurofibrillary tangles (NFT), and neuronal loss, along with a variety of other findings. Mann, Mech. Ageing Dev. 31: 213 (1985). Post-mortem slices of brain tissue of victims of AD exhibit the presence of amyloid in the form of proteinaceous extracellular cores of the neuritic plaques that are characteristic of AD. The amyloid cores of these neuritic plaques are composed of a protein called the β-amyloid (Aβ) that is arranged in a predominately beta-pleated sheet configuration.
B. Imaging of Amyloid Deposits
The first study to report human, in vivo amyloid imaging with a thioflavin derivative was presented in preliminary form by Engler et al., Neurobiol. Aging 23(1S): S429 (2002). Klunk et al., Annals of Neurology, 55: 306 (2004), provided a more detailed account. This study used a carbon-11-labeled benzothiazole derivative of the amyloid dye thioflavin-T, termed PIB (for Pittsburgh Compound-B).
As noted, the neuropathology of Alzheimer's disease frequently includes amyloid plaques, neurofibrillary tangles (Mirra et al., Neurology 41:479 (1991)), and α-synuclein deposits in the form of Lewy bodies or threads making AD a “triple amyloidosis” (Trojanowski et al., Neuromuscular Disorders 4:1 (2003)). Studies have been performed that address the relative specificity of PIB for AP amyloid deposits in light of the potential for co-deposition of NFT and α-synuclein.
At the nanomolar concentrations attainable in human Positron Emission Tomography (PET) studies, PIB and related benzothiazole derivatives bind to homogenates of plaque- and cerebrovascular amyloid-containing AD brain frontal cortex at 10-fold higher levels than the background binding observed in amyloid-free control brain frontal cortex. Klunk et al., J. Neurosci. 23: 2086 (2003).
That certain benzothiazole compounds can cross the blood brain barrier and target amyloid plaques points up a possibility of using the imaging agents to diagnose diseases associated with amyloid deposition prior to clinical symptoms. The ability to diagnose AD early and even to predict it, based on criteria seen in patients clinically diagnosed with mild cognitive impairment or another dementing disorder of questionable etiology, would enhance the care and maintenance of the elderly population afflicted with AD. To date, however, no definitive criteria have been established that would permit a physician accurately to determine onset of an amyloid deposition disease in an asymptomatic patient.
One embodiment of the present invention relates to a method of identifying a patient as prodromal to a disease associated with amyloid deposition comprising:
(A) administering to the patient, who is presenting with signs of clinical dementia or clinical signs of a mild cognitive impairment, a compound of the following formula:
then
(B) imaging said patient to obtain data; and
(C) analyzing said data to ascertain amyloid levels in said patient with reference to a normative level, thereby identifying said patient as prodromal to a disease associated with amyloid deposition. In one aspect of the invention, the patient is diagnosed with mild cognitive impairment. In another aspect of the invention, the amyloid disease is Alzheimer's disease.
The detectable label includes any atom or moiety which can be detected using an imaging technique known to those skilled in the art. Typically, the detectable label is selected from the group consisting of 3H, 131I, 125I, 123I, 76Br, 75Br, 18F, CH2—CH2—X*, O—CH2—CH2—X*, CH2—CH2—CH2—X*, O—CH2—CH2—CH2—X* (wherein X*=131I, 123I, 76Br, 75Br or 18F), 19F, 125I, a carbon-containing substituent selected from the group consisting of lower alkyl, (CH2)nOR′, CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, (C═O)N(R′)2, O(CO)R′, COOR′, CR′═CR′—Rph and CR2′—CR2′—Rph wherein at least one carbon is 11C, 13C or 14C and a chelating group (with chelated metal group) of the form W-L* or V—W-L*, wherein V is selected from the group consisting of —COO—, —CO—, —CH2O— and —CH2NH—; W is —(CH2)n where n=0, 1, 2, 3, 4, or 5; and L* is:
wherein M* is 99mTc. In a preferred embodiment, the detectable label is a radiolabel.
Using the same protocol, one can compare data obtained from the imaging techniques applied to the patients in order to:
define a dementing disorder of questionable etiology as being caused by an amyloid deposition disease;
distinguish Alzheimer's disease from frontotemporal dementia;
monitor a patient to determine onset of Alzheimer's disease;
diagnose Alzheimer's disease in a patient clinically diagnosed with mild cognitive impairment;
identify a patient as prodromal to Alzheimer's disease;
identify a patient as having a disease associated with an amyloid deposition disorder wherein the patient is presenting with a dementing disorder of questionable etiology or
identify a patient as having Alzheimer's disease wherein the patient is presenting with a dementing disorder of questionable etiology.
In one embodiment, the imaging of the inventive methodology is selected from the group consisting of gamma imaging, magnetic resonance imaging and magnetic resonance spectroscopy. In one aspect of this embodiment, the imaging is done by gamma imaging, and the gamma imaging is PET or SPECT.
In a preferred embodiment, the compound of Formula (I) is:
In particular, the above compounds contains a C-11 label.
The invention also provide methodology for identifying a patient as prodromal to a disease associated with amyloid deposition or presenting with a dementing disorder of questionable etiology previously undiagnosed with AD.
In a preferred embodiment, the amyloid deposition disorder is an amyloid plaque deposition disorder.
The present inventors have determined that certain thioflavin compounds can be used to image amyloid deposits in the brains of patients who do not meet criteria for the diagnosis of AD, such as patients presenting with clinical signs of dementia or patients with a mild cognitive impairment, including patients presenting a dementing disorder of questionable etiology, where data from amyloid imaging of patients reveals that certain amyloid deposits are a premonitory symptom of AD or another amyloid deposition disorder.
The present invention is directed to a method of identifying a patient as prodromal to a standard clinical diagnosis of a amyloid deposition disease. The method involves the use of amyloid imaging agents to obtain quantitative and qualitative data from a patient. Quantitative and qualitative amyloid imaging, in accordance with the present invention, should allow for earlier and more accurate diagnosis of amyloid deposit diseases, and should aid in the development of anti-amyloid therapies. The target patient for this methodology is a patient presenting signs of clinical dementia or a patient exhibiting clinical signs of mild cognitive impairment.
One skilled in the art would recognize that the practitioner may apply different criteria for a determination of signs of clinical dementia. Such criteria include, but are not limited to Diagnostic and Statistical Manual of Mental Disorders, third edition (DSM-III) Alzheimer's Disease Diagnostic and Treatment Center (ADDTC), International Statistical Classification of Diseases, 10th Revision (ICD-10), National Institute of Neurological Disorders and Stroke-Association Internationale pour 1a Recherche et l'Enseignment en Neurosciences (NINDS-AIREN) and Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). See Pohjasvaara et al., Stroke, 2000 31; 2952-2957.
Clinical characterization of a patient as mild cognitive impairment is well within the skill of the practitioner. Such testing of a patient to elucidate such a condition involves performing a series of mental tests. The methods for clinical diagnosis are widely reviewed and are discussed in, e.g., Petersen et al., Arch. Neurol. Vol. 56, p 303-308, March 1999.
Based on clinical testing alone, subjects identified with MCI may convert to a diagnosis of AD (at a rate of about 10-15% per year), remain MC1, or revert to a diagnosis of “normal” (10-15% per year).
Larrieu, S, Letenneur, L, Orgogozo, J M, Fabrigoule, C, Amieva, H, Le, C, Barberger-Gateau, P, Dartigues, J F (1926) Incidence and outcome of mild cognitive impairment in a population-based prospective cohort. Neurology. 59:1594-1599.
Therefore, there is considerable prognostic uncertainty associated with this clinical diagnosis. The ability to identify the presence or absence of brain amyloid deposition in a subject clinically diagnosed with MCI has the potential to greatly increase the accuracy of prognosis for conversion to AD.
The category of diseases associated with amyloid deposition includes but is not limited to Alzheimer's Disease, Down's Syndrome, Type 2 diabetes mellitus, hereditary cerebral hemorrhage amyloidosis (Dutch), amyloid A (reactive), secondary amyloidosis, familial Mediterranean fever, familial amyloid nephropathy with urticaria and deafness (Muckle-wells Syndrome), amyloid lambda L-chain or amyloid kappa L-chain (idiopathic, myeloma or macroglobulinemia-associated) A beta 2M (chronic hemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese, Japanese, Swedish)), familial amyloid cardiomyopathy (Danish), isolated cardiac amyloid, systemic senile amyloidoses, AIAPP or amylin insulinoma, atrial naturetic factor (isolated atrial amyloid), procalcitonin (medullary carcinoma of the thyroid), gelsolin (familial amyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage with amyloidosis (Icelandic)), AApo-A-I (familial amyloidotic polyneuropathy-Iowa), AApo-A-II (accelerated senescence in mice), fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, Creutzfeld Jacob disease, Gertsmann-Straussler-Scheinker syndrome, bovine spongiform encephalitis) or in cases of persons who are homozygous for the apolipoprotein E4 allele, and the condition associated with homozygosity for the apolipoprotein E4 allele or Huntington's disease. Preferably the disease associated with amyloid deposition is a amyloid plaque deposition disease. Preferably, the disease associated with amyloid deposition is AD.
According to the invention, a basic methodology of identifying a patient as prodromal to an amyloid deposition disease entails:
(A) administering to the patient, who is presenting with signs of clinical dementia or presenting with clinical signs of a mild cognitive impairment, in need thereof an effective amount of compound of the following formula:
wherein
(i) Z is S, NR′, O or C(R′)2, such that when Z is C(R′)2, the tautomeric form of the heterocyclic ring may form an indole:
wherein R′ is H or a lower alkyl group,
(ii) Y is NR1R2, OR2, or SR2,
(iii) R1 is selected from the group consisting of H, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, CH2—CH2—CH2X (wherein X═F, Cl, Br or I), (C═O)—R′, Rph, and (CH2)nRph (wherein n=1, 2, 3, or 4 and Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined below for R3-R10 and R1 is H or a lower alkyl group);
(iv) R2 is selected from the group consisting of H, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, CH2—CH2—CH2X (wherein X═F, Cl, Br or I), (C═O)—R′, Rph, and (CH2)nRph (wherein n=1, 2, 3, or 4 and Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined below for R3-R10 and R′ is H or a lower alkyl group);
(v) R3 is selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined for R1-R10 and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;
(vi) R4 is selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined for R1-R10 and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;
(vii) R5 is selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined for R1-R10 and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;
(viii) R6 is selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined for R1-R10 and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;
(ix) R7 is selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined for R1-R10 and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;
(x) R8 is selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined for R1-R10 and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;
(xi) R9 is selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined for R1-R10 and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;
(xii) R10 is selected from the group consisting of H, F, Cl, Br, I, a lower alkyl group, (CH2)nOR′ (wherein n=1, 2, or 3), CF3, CH2—CH2X, O—CH2—CH2X, CH2—CH2—CH2X, O—CH2—CH2—CH2X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, N(R′)2, NO2, (C═O)N(R′)2, O(CO)R′, OR′, SR′, COOR′, Rph, CR′═CR′—Rph, CR2′—CR2′—Rph (wherein Rph represents an unsubstituted or substituted phenyl group with the phenyl substituents being chosen from any of the non-phenyl substituents defined for R1-R10 and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;
alternatively, one of R3-R10 may be a chelating group (with or without a chelated metal group) of the form W-L or V—W-L, wherein V is selected from the group consisting of —COO—, —CO—, —CH2O— and —CH2NH—; W is —(CH2)n where n=0, 1, 2, 3, 4, or 5; and L is:
wherein M is selected from the group consisting of Tc and Re;
and radiolabeled derivatives and pharmaceutically acceptable salts thereof, where at least one of the substituent moieties comprises a detectable label;
(B) imaging said patient to obtain data and
(C) analyzing said data to ascertain amyloid levels in said patient with reference to a normative patient.
One embodiment relates to a method for diagnosing a patient presenting with a dementing disorder of questionable etiology. This method would involve determining if dementias of questionable etiology are likely to be AD or another amyloid deposition disorder based on the finding of amyloid deposition. This method would involve administering to a patient a compound of Formula (I) or (II) or one of structures 1-45, imaging the patient to obtain data and determining if the dementia of questionable etiology is AD based on the finding of amyloid deposition.
Another embodiment is a method of manufacturing a medicament for identifying a patient as prodromal to an amyloid deposition disease as described in any of the foregoing or following embodiments. The method comprises combining a compound according to formula I or II or one of structures 1-45 described herein, with a pharmaceutical carrier to form the medicament.
Yet another embodiment is a method of manufacturing a medicament for diagnosing a patient presenting with a dementing disorder of questionable etiology as set forth in any of the foregoing or following embodiments. The method comprises combining a compound according to formula I or II or one of structures 1-45 described herein, with a pharmaceutical carrier to form the medicament.
The term “dementing disorder of questionable etiology” refers to the condition in which a person presents for clinical evaluation (which may consist of neurological, psychiatric, medical and neuropsychological evaluations commonly employed by those skilled in the art of diagnosing persons with dementing disorders) and, after that clinical evaluation, the evaluator finds evidence that some dementing disorder may be present (based on evidence of subjective memory complaints, description of memory complaints by informants familiar with the persons deviation from normal functioning, or poor performance on neuropsychological and clinical tests commonly used by those skilled in the art), but, can not find sufficient evidence for any single clinically defined dementing disorder (such as AD, frontotemporal dementia, Dementia with Lewy Bodies, Vascular dementia, pseudodementia due to Major Depression, Creutzfeld Jacob disease and others known to those skilled in the art) or finds that the person shows evidence of more than one single dementing disorder to the degree that the distinction between these two (or more) dementing disorders is questionable in this person.
This aspect of the invention employs amyloid imaging agents which, in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), are used to quantify amyloid deposition in vivo. These imaging techniques acquire data on many brain regions. Quantitation on specific regions is achieved by delineating “regions of interest or ROI”.
Pursuant to the invention, data obtained from patients using one of the imaging techniques mentioned above can be compared to data from normative patients with a conclusion based on criteria which distinguish the patient as prodromal to a standard clinical diagnosis of an amyloid deposition disease.
Using the same protocol, one can compare data obtained from the imaging techniques applied to the patients in order to:
define a dementing disorder of questionable etiology as being caused by an amyloid deposition disease;
distinguish Alzheimer's disease from frontotemporal dementia;
monitor a patient to determine onset of Alzheimer's disease;
diagnose Alzheimer's disease in a patient clinically diagnosed with mild cognitive impairment;
identify a patient as prodromal to Alzheimer's disease;
identify a patient as having a disease associated with an amyloid deposition disorder wherein the patient is presenting with a dementing disorder of questionable etiology or identify a patient as having Alzheimer's disease wherein the patient is presenting with a dementing disorder of questionable etiology.
An amyloid imaging agent suitable for the present invention is any compound of formula (I), described above.
In some embodiments, the amyloid imaging agent is a compound of formula (II)
or a radiolabeled derivative, pharmaceutically acceptable salt, hydrate, solvate or prodrug of the compound, wherein:
R1 is hydrogen, —OH, —NO2, —CN, —COOR, —OCH2OR, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy or halo;
R is C1-C6 alkyl;
R2 is hydrogen or halo;
R3 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; and
R4 is hydrogen, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, wherein the alkyl, alkenyl or alkynyl comprises a radioactive carbon or is substituted with a radioactive halo when R2 is hydrogen or a non-radioactive halo;
provided that when R1 is hydrogen or —OH, R2 is hydrogen and R4 is —11CH3, then R3 is C2-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl; and
further provided that when R1 is hydrogen, R2 hydrogen and R4 is —(CH2)318F, then R3 is C2-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl.
In one embodiment, R2 in the compounds of formula (II) contains a radioactive halo. Thus, for example, one compound of formula (II) for use in combination with any of the embodiments described herein is 2-(3-18F-Fluoro-4-methylamino-phenyl)-benzothiazol-6-ol:
“Alkyl” refers to a saturated straight or branched chain hydrocarbon radical. Examples include without limitation methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl and n-hexyl. The term “lower alkyl” refers to C1-C6 alkyl.
“Alkenyl” refers to an unsaturated straight or branched chain hydrocarbon radical comprising at least one carbon to carbon double bond. Examples include without limitation ethenyl, propenyl, iso-propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyl and n-hexenyl.
“Alkynyl” refers to an unsaturated straight or branched chain hydrocarbon radical comprising at least one carbon to carbon triple bond. Examples include without limitation ethynyl, propynyl, iso-propynyl, butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl.
“Alkoxy” refers to an alkyl group bonded through an oxygen linkage.
“Halo” refers to a fluoro, chloro, bromo or iodo radical.
“Radioactive halo” refers to a radioactive halo, i.e. radiofluoro, radiochloro, radiobromo or radioiodo.
In another embodiment, the thioflavin compound of formula (I) is selected from the group consisting of structures 1-45 or a radiolabeled derivative thereof:
In the compounds I-45, at least one of the substituent moieties comprises a detectable label as defined above.
In preferred embodiments, the amyloid imaging agent is {N-methyl-11C}2-[4′-(methylamino)phenyl]6-hydroxybenzothiazole (“[11C]PIB”) or {N-methyl-3H}2-[4′-(methylamino)phenyl]6-hydroxybenzothiazole (“[3H]PIB”).
“Effective amount” refers to the amount required to produce a desired effect. Examples of an “effective amount” include amounts that enable detecting and imaging of amyloid deposit(s) in vivo or in vitro, that yield acceptable toxicity and bioavailability levels for pharmaceutical use, and/or prevent cell degeneration and toxicity associated with fibril formation.
Compounds of formulas (I) and (II) or structures 1-45, also referred to herein as “thioflavin compounds,” “thioflavin derivatives,” or “amyloid imaging agents,” have each of the following characteristics: (1) specific binding to synthetic AP in vitro and (2) ability to cross a non-compromised blood brain barrier in vivo.
The thioflavin compounds and radiolabeled derivatives thereof of formulas (I) (II) and structures 1-45 cross the blood brain barrier in vivo and bind to AP deposited in neuritic (but not diffuse) plaques, to AP deposited in cerebrovascular amyloid, and to the amyloid consisting of the protein deposited in NFT. The present compounds are non-quaternary amine derivatives of Thioflavin S and T which are known to stain amyloid in tissue sections and bind to synthetic AP in vitro. Kelenyi J. Histochem. Cytochem. 15: 172 (1967); Burns et al. J Path. Bact. 94:337 (1967); Guntern et al. Experientia 48: 8 (1992); LeVine Meth. Enzymol. 309: 274 (1999).
The method of this invention determines the presence and location of amyloid deposits in an organ or body area, preferably brain, of a patient. The present method comprises administration of a detectable quantity of an amyloid imaging agent of formulas (I) or (II). In some embodiments, the amyloid imaging agent is chosen from structures 1-45, as shown above. An amyloid imaging agent may be administered to a patient as a pharmaceutical composition or a pharmaceutically acceptable water-soluble salt thereof.
“Pharmaceutically acceptable salt” refers to an acid or base salt of the inventive compound, which salt possesses the desired pharmacological activity and is neither biologically nor otherwise undesirable. The salt can be formed with acids that include without limitation acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride hydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate, tosylate and undecanoate. Examples of a base salt include without limitation ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine and lysine. In some embodiments, the basic nitrogen-containing groups can be quarternized with agents including lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides such as phenethyl bromides.
Generally, the dosage of the detectably labeled thioflavin derivative will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, concomitant therapies and other variables, to be adjusted by a physician skilled in the art. Dosage can vary from 0.001 μg/kg to 10 μg/kg, preferably 0.01 μg/kg to 1.0 μg/kg.
Administration to the subject may be local or systemic and accomplished intravenously, intraarterially, intrathecally (via the spinal fluid) or the like. Administration may also be intradermal or intracavitary, depending upon the body site under examination. After a sufficient time has elapsed for the compound to bind with the amyloid, for example 30 minutes to 48 hours, the area of the subject under investigation is examined by routine imaging techniques such as MRS/MRI, SPECT, planar scintillation imaging, PET, and any emerging imaging techniques, as well. The exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan. For brain imaging, preferably, the amount (total or specific binding) of the bound radioactively labeled thioflavin derivative or analogue of the present invention is measured and compared (as a ratio) with the amount of labeled thioflavin derivative bound to the cerebellum of the patient. This ratio is then compared to the same ratio in age-matched normal brain.
The amyloid imaging agents of the present invention are advantageously administered in the form of injectable compositions, but may also be formulated into well known drug delivery systems (e.g., oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), intracisternal, intravaginal, intraperitoneal, local (powders, ointments or drops), or as a buccal or nasal spray). A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain about 10 mg of human serum albumin and from about 0.5 to 500 micrograms of the labeled thioflavin derivative per milliliter of phosphate buffer containing NaCl. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington: American Pharmaceutical Association (1975), the contents of which are hereby incorporated by reference.
Particularly preferred amyloid imaging agents of the present invention are those that, in addition to specifically binding amyloid in vivo and capable of crossing the blood brain barrier, are also non-toxic at appropriate dosage levels and have a satisfactory duration of effect.
According to the present invention, a pharmaceutical composition comprising an amyloid imaging agent of formula (I) or (II) or structures 1-45, is administered to subjects in whom amyloid or amyloid fibril formation are anticipated, e.g., patients clinically diagnosed with Alzheimer's disease.
Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to routine skills in the art. See, Goodman and Gilman's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).
The invention employs amyloid imaging agents which, in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imaging such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), are used to quantify amyloid deposition in vivo. These imaging techniques acquire data on many brain regions. Quantitation on specific regions is achieved by delineating “regions of interest or ROI”. The method involves imaging a patient to establish amyloid deposition.
The term “in vivo imaging” refers to any method which permits the detection of a labeled thioflavin derivative of formulas (I) or (II) or structures 1-45. For gamma imaging, the radiation emitted from the organ or area being examined is measured and expressed either as total binding or as a ratio in which total binding in one tissue is normalized to (for example, divided by) the total binding in another tissue of the same subject during the same in vivo imaging procedure. Total binding in vivo is defined as the entire signal detected in a tissue by an in vivo imaging technique without the need for correction by a second injection of an identical quantity of labeled compound along with a large excess of unlabeled, but otherwise chemically identical compound. A “subject” is a mammal, preferably a human, and most preferably a human suspected of having a disease associated with amyloid deposition, such as AD and/or dementia. The term “subject” and “patient” are used interchangeably herein.
For purposes of in vivo imaging, the type of detection instrument available is a major factor in selecting a given label. For instance, radioactive isotopes and 18F are well suited for in vivo imaging in the methods of the present invention. The type of instrument used will guide the selection of the radionuclide or stable isotope. For instance, the radionuclide chosen must have a type of decay detectable by a given type of instrument. Another consideration relates to the half-life of the radionuclide. The half-life should be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that the host does not sustain deleterious radiation. The radiolabeled compounds of the invention can be detected using gamma imaging wherein emitted gamma irradiation of the appropriate wavelength is detected. Methods of gamma imaging include, but are not limited to, SPECT and PET. Preferably, for SPECT detection, the chosen radiolabel will lack a particulate emission, but will produce a large number of photons in a 140-200 keV range. For PET detection, the radiolabel will be a positron-emitting radionuclide such as 19F which will annihilate to form two 511 keV gamma rays which will be detected by the PET camera.
In the present invention, amyloid binding compounds, which are useful for in vivo imaging and quantification of amyloid deposition, are administered to a patient. These compounds are to be used in conjunction with non-invasive neuroimaging techniques such as magnetic resonance spectroscopy (MRS) or imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). In accordance with this invention, the thioflavin derivatives may be labeled with 19F or 13C for MRS/MRI by general organic chemistry techniques known to the art. See, e.g., March, J. ADVANCED ORGANIC CHEMISTRY: REACTIONS, MECHANISMS, AND STRUCTURE (3rd Edition, 1985), the contents of which are hereby incorporated by reference. The thioflavin derivatives also may be radiolabeled with 18F, 11C, 75Br, or 76Br for PET by techniques well known in the art and are described by Fowler, J. and Wolf, A. in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H. eds.) 391-450 (Raven Press, NY 1986) the contents of which are hereby incorporated by reference. The thioflavin derivatives also may be radiolabeled with 1231 for SPECT by any of several techniques known to the art. See, e.g., Kulkami, Int. J. Rad. Appl. & Inst. (Part B) 18: 647 (1991), the contents of which are hereby incorporated by reference. In addition, the thioflavin derivatives may be labeled with any suitable radioactive iodine isotope, such as, but not limited to 131I, 125I, or 123I, by iodination of a diazotized amino derivative directly via a diazonium iodide, see Greenbaum, F. Am. J. Pharm. 108: 17 (1936), or by conversion of the unstable diazotized amine to the stable triazene, or by conversion of a non-radioactive halogenated precursor to a stable tri-alkyl tin derivative which then can be converted to the iodo compound by several methods well known to the art. See, Satyamurthy and Barrio J. Org. Chem. 48: 4394 (1983), Goodman et al., J. Org. Chem. 49: 2322 (1984), and Mathis et al., J Labell. Comp. and Radiopharm. 1994: 905; Chumpradit et al., J. Med. Chem. 34: 877 (1991); Zhuang et al., J. Med. Chem. 37: 1406 (1994); Chumpradit et al., J. Med. Chem. 37: 4245 (1994). For example, a stable triazene or tri-alkyl tin derivative of thioflavin or its analogues is reacted with a halogenating agent containing 131I, 125I, 123I, 76Br, 75Br, 18F or 19F. Thus, the stable tri-alkyl tin derivatives of thioflavin and its analogues are novel precursors useful for the synthesis of many of the radiolabeled compounds within the present invention. As such, these tri-alkyl tin derivatives are one embodiment of this invention.
The thioflavin derivatives also may be radiolabeled with known metal radiolabels, such as Technetium-99m (99mTc). Modification of the substituents to introduce ligands that bind such metal ions can be effected without undue experimentation by one of ordinary skill in the radiolabeling art. The metal radiolabeled thioflavin derivative can then be used to detect amyloid deposits. Preparing radiolabeled derivatives of Tc99m is well known in the art. See, for example, Zhuang et al., “Neutral and stereospecific Tc-99m complexes: [99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT)” Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., “Small and neutral Tc(v) O BAT, bisaminoethanethiol (N2S2) complexes for developing new brain imaging agents” Nuclear Medicine & Biology 25(2):135-40, (1998); and Hom et al., “Technetium-99m-labeled receptor-specific small-molecule radiopharmaceuticals: recent developments and encouraging results” Nuclear Medicine & Biology 24(6):485-98, (1997).
The methods of the present invention may use isotopes detectable by nuclear magnetic resonance spectroscopy for purposes of in vivo imaging and spectroscopy. Elements particularly useful in magnetic resonance spectroscopy include 19F and 13C.
Suitable radioisotopes for purposes of this invention include beta-emitters, gamma-emitters, positron-emitters, and x-ray emitters. These radioisotopes include 131I, 123I, 18F, 11C, 75Br, and 76Br. Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) or Spectroscopy (MRS), according to this invention, include 19F and 13C. Suitable radioisotopes for in vitro quantification of amyloid in homogenates of biopsy or post-mortem tissue include 125I, 14C, and 3H. The preferred radiolabels are 11C or 18F for use in PET in vivo imaging, 123I for use in SPECT imaging, 19F for MRS/MRI, and 3H or 14C for in vitro studies. However, any conventional method for visualizing imaging agents can be utilized in accordance with this invention.
The ability of the compound of formulas (I) and (II) or structures 1-45 to specifically bind to amyloid plaques over neurofibrially tangles is particularly true at concentrations less than 10 nM, which includes the in vivo concentration range of PET radiotraces. At these low concentrations, in homogenates of brain tissue which contain only tangles and no plaques, significant binding does not result when compared to control brain tissue containing neither plaques nor tangles. However, incubation of homogenates of brain tissue which contains mainly plaques and some tangles with radiolabeled compounds of formulas (I) and (II) or structures 1-45, results in a significant increase in binding when compared to control tissue without plaques or tangles. This data suggests the advantage that these compounds are specific for Aβ deposits at concentrations less than 10 nM. These low concentrations are then detectable with PET studies, making PET detection using radiolabeled compounds of formulas (I) and (II) or structures 1-45 which are specific for Aβ deposits possible. The use of such compounds permits PET detection in Aβ deposits such as those found in plaques and cerebrovascular amyloid. Since it has been reported that levels of insoluble, deposited Aβ in the frontal cortex are increased prior to tangle formation, this would suggest that radiolabeled compounds of formulas (I) and (II) or structures 1-45, used as PET tracers, would be specific for the earliest changes in AD cortex. Naslund et al. JAMA 283:1571 (2000).
Unless the context clearly dictates otherwise, the definitions of singular terms may be extrapolated to apply to their plural counterparts as they appear in the application; likewise, the definitions of plural terms may be extrapolated to apply to their singular counterparts as they appear in the application.
The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. Throughout the specification, any and all references to a publicly available document, including U.S. patents, are specifically incorporated into this patent application by reference.
The data obtained can be quantitatively expressed in terms of Standardized Uptake Value (SUV) or in terms of pharmacokinetic modeling parameters such as the Logan distribution volume ratio (DVR) to a reference tissue such as cerebellum. Subjects who are more than one standard deviation above the typical control value of SUV or DVR would be considered to have a “positive” test and be considered to be prodromal to a clinical diagnosis of an amyloid deposition disease such as AD. Specifically, subjects will be considered “positive” if their 40-60 min average SUV is greater than 1.0 in frontal, parietal or posterior cingulate cortex. This value clearly separated AD patients from controls in the initial human study (Klunk, et al., 2004, Ann. Neurol., 55(3):306-19) (see
Compounds of formulas (I) and (II), and the formulae of structures 1-45, can be prepared by methods that are well known in the art. See, e.g., WO 02/16333 and U.S. Patent Publication No. 2003/0236391, published Dec. 25, 2003, the entire contents of which are herein incorporated by reference.
All of the reagents used in the synthesis were purchased from Aldrich Chemical Company and used without further purification, unless otherwise indicated. Melting points were determined on MeI-TEMP II and were uncorrected. The 1H NMR spectra of all compounds were measured on Bruker 300 using TMS as internal reference and were in agreement with the assigned structures. The TLC was performed using Silica Gel 60 F254 from EM Sciences and detected under UV lamp.
Flash chromatography was performed on silica gel 60 (230-400 mesh. Purchased from Mallinckrodt Company. The reverse phase TLC were purchased from Whiteman Company.
R1 is hydrogen, —OH, —NO2, —CN, —COOR, —OCH2OR, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy or halo, wherein one or more of the atoms of R1 may be a radiolabeled atom;
R is C1-C6 alkyl, wherein one or more of the carbon atoms may be a radiolabeled atom;
is hydrolysed by one of the following two procedures:
The 6-substituted 2-aminobenzothiazole (172 mmol) is suspended in 50% KOH (180 g KOH dissolved in 180 mL water) and ethylene glycol (40 mL). The suspension is heated to reflux for 48 hours. Upon cooling to room temperature, toluene (300 mL) is added and the reaction mixture is neutralized with acetic acid (180 mL). The organic layer is separated and the aqueous layer is extracted with another 200 mL of toluene. The toluene layers are combined and washed with water and dried over MgSO4. Evaporation of the solvent gives the desired product.
The 6-substituted-benzothiazole (6.7 mmol) is suspended in ethanol (11 mL, anhydrous) and hydrazine (2.4 mL) is added under a nitrogen atmosphere at room temperature. The reaction mixture is heated to reflux for 1 hour. The solvent is evaporated and the residue is dissolved into water (10 mL) and adjusted to a pH of 5 with acetic acid. The precipitate is collected with filtration and washed with water to give the desired product.
The resulting 5-substituted-2-amino-1-thiophenol of the form
is coupled to a benzoic acid of the form:
wherein R2 is hydrogen, and R3 and R4 are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl
by the following methodology:
A mixture of the 5-substituted 2-aminothiophenol (4.0 mmol), the benzoic acid (4.0 mmol), and polyphosphoric acid (PPA) (10 g) is heated to 220° C. for 4 hours. The reaction mixture is cooled to room temperature and poured into 10% potassium carbonate solution (˜400 mL). The precipitate is collected by filtration under reduced pressure to give the desired product, which can be purified by flash chromatography or recrystallization.
The R2 hydrogen can be substituted with either a non-radioactive halo or a radioactive halo by the following reaction:
To a solution of 6-substituted 2-(4′-aminophenyl)-benzothiazole (1 mg) in 250 μL acetic acid in a sealed vial is added 40 μL of chloramine-T solution (28 mg dissolved in 500 μL acetic acid) followed by 27 μL (ca. 5 mCi) of sodium [125I]iodide (specific activity 2,175 Ci/mmol). The reaction mixture is stirred at room temperature for 2.5 hours and quenched with saturated sodium hydrogensulfite solution. After dilution with 20 ml of water, the reaction mixture is loaded onto C8 Plus SepPak and eluted with 2 ml methanol. Depending on the nature of the substituent on the 6-position, protecting groups may need to be employed. For example, the 6-hydroxy group is protected as the methanesulfonyl(mesyloxy) derivative. For deprotection of the methanesulfonyl group, 0.5 ml of 1 M NaOH is added to the eluted solution of radioiodinated intermediate. The mixture is heated at 50° C. for 2 hours. After being quenched by 500 μL of 1 M acetic acid, the reaction mixture is diluted with 40 mL of water and loaded onto a C8 Plus SepPak. The radioiodinated product, having a radioactivity of ca. 3 mCi, is eluted off the SepPak with 2 mL of methanol. The solution is condensed by a nitrogen stream to 300 μL and the crude product is purified by HPLC on a Phenomenex ODS column (MeCN/TEA buffer, 35:65, pH 7.5, flow rate 0.5 mL/minute up to 4 minutes, 1.0 mL/minute at 4-6 minutes, and 2.0 mL/minute after 6 minutes, retention time 23.6). The collected fractions are loaded onto a C8 Plus SepPak. Elution with 1 mL of ethanol gave ca. 1 mCi of the final radioiodinated product.
When either or both R3 and R4 are hydrogen, then R3 and R4 can be converted to C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl by reaction with an alkyl, alkenyl or alkynyl halide under the following conditions:
For dialkylation: To a solution of 6-substituted 2-(4′-aminophenyl)-benzothiazole (0.59 mmol) in DMSO (anhydrous, 2 ml) are added alkyl, alkenyl, or alkynyl halide (2.09 mmol), and K2CO3 (500 mg, 3.75 mmol). The reaction mixture is heated at 140° C. for 16 hours. Upon cooling to room temperature, the reaction mixture is poured into water and extracted with ethyl acetate (3×10 mL). The organic layers are combined and the solvent is evaporated. The residue is purified by flash column to give the desired 6-substituted dimethylaminophenyl)-benzothiazole.
For monoalkylation: To a solution of 6-substituted 2-(4′-aminophenyl)benzothiazole (0.013 mmol) in DMSO (anhydrous, 0.5 ml) is added alkyl, alkenyl, or alkynyl halide (0.027 mmol) and anhydrous K2CO3 (100 mg, 0.75 mmol). The reaction mixture is heated at 100° C. for 16 hours. Upon cooling to room temperature, the reaction mixture is directly purified by normal phase preparative TLC to give the desired 6-substituted-2-(4′-methylaminophenyl)-benzothiazole derivatives.
When R2 is hydrogen or a non-radioactive halo, R4 is C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl, wherein the alkyl, alkenyl or alkynyl comprises a radioactive carbon or is substituted with a radioactive halo, the compound can be synthesized by one of the following sequences:
Approximately 1 Ci of [11C]carbon dioxide is produced using a CTI/Siemens RDS 112 negative ion cyclotron by irradiation of a nitrogen gas (14N2) target containing 1% oxygen gas with a 40 μA beam current of 11 MeV protons for 60 minutes. [11C]Carbon dioxide is converted to [11C]methyl iodide by first reacting it with a saturated solution of lithium aluminum hydride in THF followed by the addition of hydriodic acid at reflux temperature to generate [11C]methyl iodide. The [11C]methyl iodide is carried in a stream of nitrogen gas to a reaction vial containing the precursor for radiolabeling. The precursor, 6-substituted 2-(4′-aminophenyl)-benzothiazole (˜3.7 μmoles), is dissolved in 400 μL of DMSO. Dry KOH (10 mg) is added, and the 3 mL V-vial is vortexed for 5 minutes. No-carrier-added [11C]methyl iodide is bubbled through the solution at 30 mL/minute at room temperature. The reaction is heated for 5 minutes at 95° C. using an oil bath. The reaction product is purified by semi-preparative HPLC using a Prodigy ODS-Prep column eluted with 60% acetonitrile/40% triethylammonium phosphate buffer pH 7.2 (flow at 5 mL/minute for 0-7 minutes then increased to 15 mL/minute for 7-30 minutes). The fraction containing [N-methyl-11C]6-substituted 2-(4′-methylaminophenyl)-benzothiazole (at about 15 min) is collected and diluted with 50 mL of water and eluted through a Waters C18 SepPak Plus cartridge. The C18 SepPak is washed with 10 mL of water, and the product is eluted with 1 mL of ethanol (absolute) into a sterile vial followed by 14 mL of saline. Radiochemical and chemical purities are >95% as determined by analytical HPLC (k′=4.4 using the Prodigy ODS(3) analytical column eluted with 65/35 acetonitrile/triethylammonium phosphate buffer pH 7.2). The radiochemical yield averages 17% at EOS based on [11C]methyl iodide, and the specific activity averages about 160 GBq/μmol (4.3 Ci/μmol) at end of synthesis.
For radioactive Halogen Incorporation:
A mixture of 6-substituted 2-(4′-aminophenyl)-benzathiazole (protecting groups may be necessary depending on the nature of the 6-substituent as noted above) (0.22 mmol), NaH (4.2 mmol) and 2-(-3-bromopropoxy)tetrahydro-2-H-pyran (0.22 mmol) in THF (8 mL) is heated to reflux for 23 hours. The solvent is removed by distillation and the residue is dissolved in to ethyl acetate and water, the organic layer is separated and the aqueous layer is extracted with ethyl acetate (10 mL×6). The organic layer is combined and dried over MgSO4 and evaporated to dryness. The residue is added AcOH/THF/H2O solution (5 mL, 4/2/1) and heated to 10° C. for 4 hours. The solvent is removed by evaporation and the residue is dissolved in ethyl acetate (˜10 mL) washed by NaHCO3 solution, dried over MgSO4 and evaporated to dryness to give a residue which is purified with preparative TLC (hexane:ethyl acetate=60:40) to give the desired 6-substituted 2-(4′-(3″-hydroxypropylamino)-phenyl)-benzothiazole (45%).
To a solution of 6-substituted 2-(4′-(3″-hydroxypropylamino)-phenyl)-benzathiazole (0.052 mmol) and Et3N (0.5 ml) dissolved in acetone (5 mL) is added (BOC)2O (50 mg, 0.22 mmol). The reaction mixture is stirred at room temperature for 6 hours followed by addition of tosyl chloride (20 mg, 0.11 mmol). The reaction mixture is stirred at room temperature for another 24 hours. The solvent is removed and the residue is dissolved into ethyl acetate (10 mL), washed with NaCO3 solution, dried over MgSO4, evaporated, and purified with flash column (Hexane/ethyl acetate=4/1) to give the desired 6-substituted 2-(4′-(3″-toluenesulfonoxypropylamino)-phenyl)-benzothiazole (13%). This 6-substituted 2-(4′-(3″-toluenesulfonoxypropylamino)-phenyl)-benzothiazole is then radiofluorinated by standard methods as follows:
A cyclotron target containing 0.35 mL of 95% [O-18]-enriched water is irradiated with 11 MeV protons at 20 μA of beam current for 60 minutes, and the contents are transferred to a 5 mL reaction vial containing Kryptofix 222 (22.3 mg) and K2CO3 (7.9 mg) in acetonitrile (57 μL). The solution is evaporated to dryness three times at 110° C. under a stream of argon following the addition of 1 mL aliquots of acetonitrile. To the dried [F-18]fluoride is added 3 mg of 6-substituted 2-(4′-(3″-toluenesulfonoxypropylamino)-phenyl)-benzothiazole in 1 mL DMSO, and the reaction vial is sealed and heated to 85° C. for 30 minutes. To the reaction vial, 0.5 mL of MeOH/HCl (concentrated) (2/1 v/v) is added, and the vial is heated at 120° C. for 10 minutes. After heating, 0.3 mL of 2 M sodium acetate buffer is added to the reaction solution followed by purification by semi-prep HPLC using a Phenomenex Prodigy ODS-prep C18 column (10 μm 250×10 mm) eluted with 40% acetonitrile/60% 60 mM triethylamine-phosphate buffer (v/v) pH 7.2 at a flow rate of 5 mL/minute for 15 minutes, then the flow is increased to 8 mL/minute for the remainder of the separation. The product, [F-18]6-substituted 2-(4′-(3″-fluoropropylamino)-phenyl)-benzothiazole, is eluted at 20 minutes in a volume of about 16 mL. The fraction containing [F-18]6-substituted 2-(4′-(3″-fluoropropylamino)-phenyl)-benzothiazole is diluted with 50 mL of water and eluted through a Waters C18 SepPak Plus cartridge. The SepPak cartridge is then washed with 10 mL of water, and the product is eluted using 1 mL of ethanol (absol.) into a sterile vial. The solution is diluted with 10 mL of sterile normal saline for intravenous injection into animals. The [F-18]6-substituted 2-(4′-(3″-fluoropropylamino)-phenyl)-benzothiazole product is obtained in 2-12% radiochemical yield at the end of the 120 minute radiosynthesis (not decay corrected) with an average specific activity of 1500 Ci/mmol.
Approximately 1 Ci of [11C]carbon dioxide was produced using a CTI/Siemens RDS 112 negative ion cyclotron by irradiation of a nitrogen gas (14N2) target containing 1% oxygen gas with a 40 μA beam current of 11 MeV protons for 60 minutes. [11C]Carbon dioxide is converted to [11C]methyl iodide by first reacting it with a saturated solution of lithium aluminum hydride in THF followed by the addition of hydriodic acid at reflux temperature to generate [11C]methyl iodide. The [11C]methyl iodide is carried in stream of nitrogen gas to a reaction vial containing the precursor for radiolabeling. The precursor, 6-CH3O-BTA-1 (1.0 mg, 3.7 μmoles), was dissolved in 400 μL of DMSO. Dry KOH (10 mg) was added, and the 3 mL V-vial was vortexed for 5 minutes. No-carrier-added [11C]methyl iodide was bubbled through the solution at 30 mL/minute at room temperature. The reaction was heated for 5 minutes at 95° C. using an oil bath. The reaction product was purified by semi-preparative HPLC using a Prodigy ODS-Prep column eluted with 60% acetonitrile/40% triethylammonium phosphate buffer pH 7.2 (flow at 5 mL/minute for 0-7 minutes then increased to 15 mL/minute for 7-30 minutes). The fraction containing [N-Methyl-11C]2-(4′-Dimethylaminophenyl)-6-methoxy-benzothiazole (at about 15 minutes) was collected and diluted with 50 mL of water and eluted through a Waters C18 SepPak Plus cartridge. The C18 SepPak was washed with 10 mL of water, and the product was eluted with 1 mL of ethanol (absolute) into a sterile vial followed by 14 mL of saline. Radiochemical and chemical purities were >95% as determined by analytical HPLC (k′=4.4 using the Prodigy ODS(3) analytical column eluted with 65/35 acetonitrile/triethylammonium phosphate buffer pH 7.2). The radiochemical yield averaged 17% at EOS based on [11C]methyl iodide, and the specific activity averaged about 160 GBq/μmol (4.3 Ci/μmol) at end of synthesis.
To a solution of 2-(4′-aminophenyl)-6-methanesulfonoxy-benzothiazole (1 mg) in 250 μL acetic acid in a sealed vial was added 40 μL of chloramine T solution (28 mg dissolved in 500 μL acetic acid) followed by 27 μL (ca. 5 mCi) of sodium [125I]iodide (specific activity 2,175 Ci/mmol). The reaction mixture was stirred at room temperature for 2.5 hours and quenched with saturated sodium hydrogensulfite solution. After dilution with 20 ml of water, the reaction mixture was loaded onto C8 Plus SepPak and eluted with 2 ml methanol. For deprotection of the methanesulfonyl group, 0.5 ml of 1 M NaOH was added to the eluted solution of radioiodinated intermediate. The mixture was heated at 50° C. for 2 hours. After being quenched by 500 μL of 1 M acetic acid, the reaction mixture was diluted with 40 mL of water and loaded onto a C8 Plus SepPak. The radioiodinated product, having a radioactivity of ca. 3 mCi, was eluted off the SepPak with 2 mL of methanol. The solution was condensed by a nitrogen stream to 300 μL and the crude product was purified by HPLC on a Phenomenex ODS column (MeCN/TEA buffer, 35:65, pH 7.5, flow rate 0.5 mL/minute up to 4 minutes, 1.0 mL/minute at 4-6 minutes, and 2.0 mL/minute after 6 minutes, retention time 23.6). The collected fractions were loaded onto a C8 Plus SepPak. Elution with 1 mL of ethanol gave ca. 1 mCi of the final radioiodinated product.
Preparation of the 123I radiolabeled derivatives, proceeds similarly to the synthesis outlined above. For example, replacing sodium [125I]iodide with sodium [123I]iodide in the synthetic method would provide the 1231 radiolabeled compound. Such substitution of one radiohalo atom for another is well known in the art, see for example, Mathis C A, Taylor S E, Biegon A, Enas J D. [125I]5-Iodo-6-nitroquipazine: a potent and selective ligand for the 5-hydroxytryptamine uptake complex I. In vitro studies. Brain Research 1993; 619:229-235; Jagust W, Eberling J L, Roberts J A, Brennan K M, Hanrahan S M, Van Brocklin H, Biegon A, Mathis C A. In vivo imaging of the 5-hydroxytryptamine reuptake site in primate brain using SPECT and [123I]5-iodo-6-nitroquipazine. European Journal of Pharmacology 1993; 242:189-193; Jagust W J, Eberling J L, Biegon A, Taylor S E, VanBrocklin H, Jordan S, Hanrahan S M, Roberts J A, Brennan K M, Mathis C A. [Iodine-123]5-Iodo-6-Nitroquipazine: SPECT Radiotracer to Image the Serotonin Transporter. Journal of Nuclear Medicine 1996; 37:1207-1214.)
A cyclotron target containing 0.35 mL of 95% [0-18]-enriched water was irradiated with 11 MeV protons at 20 μA of beam current for 60 minutes, and the contents were transferred to a 5 mL reaction vial containing 2 mg Cs2CO3 in acetonitrile (57 μL). The solution was evaporated to dryness at 110° C. under a stream of argon three times using 1 mL aliquots of acetonitrile. To the dried [F-18]fluoride was added 6 mg of 6-MOMO-BT-3′-Cl-4′-NO2 in 1 mL DMSO, and the reaction vial was sealed and heated to 120° C. for 20 minutes (radiochemical incorporation for this first radiosynthesis step was about 20% of solubilized [F-18]fluoride). To the crude reaction mixture was added 8 mL of water and 6 mL of diethyl ether, the mixture was shaken and allowed to separate. The ether phase was removed and evaporated to dryness under a stream of argon at 120° C. To the dried sample, 0.5 mL of absolute EtOH was added along with 3 mg copper (II) acetate and 8 mg of NaBH4. The reduction reaction was allowed to proceed for 10 minutes at room temperature (the crude yield for the reduction step was about 40%). To the reaction mixture was added 8 mL of water and 6 mL of diethyl ether, the mixture was shaken and the ether phase separated. The diethyl ether phase was dried under a stream of argon at 120° C. To the reaction vial, 700 uL of DMSO was added containing 30 micromoles of CH3I and 20 mg of dry KOH. The reaction vial was heated at 120° C. for 10 minutes. A solution of 700 uL of 2:1 MeOH/HCl (concentrated) was added and heated for 15 minutes at 120° C. After heating, 1 mL of 2 M sodium acetate buffer was added to the reaction solution followed by purification by semi-prep HPLC using a Phenomenex Prodigy ODS-prep C18 column (10 μm 250×10 mm) eluted with 35% acetonitrile/65% 60 mM triethylamine-phosphate buffer (v/v) pH 7.2 at a flow rate of 5 mL/minute for 2 minutes, then the flow was increased to 15 mL/minute for the remainder of the separation. The product, 2-(3-18F-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol, eluted at ˜15 minutes in a volume of about 16 mL. The fraction containing 2-(3-18F-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol was diluted with 50 mL of water and eluted through a Waters C18 SepPak Plus cartridge. The SepPak cartridge was then washed with 10 mL of water, and the product was eluted using 1 mL of ethanol (absol.) into a sterile vial. The solution was diluted with 10 mL of sterile normal saline for intravenous injection into animals. The 2-(3−18F-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol product was obtained in 0.5% (n=4) radiochemical yield at the end of the 120 minute radiosynthesis (not decay corrected) with an average specific activity of 1000 Ci/mmol. The radiochemical and chemical purities of 2-(3-18F-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol were assessed by radio-HPLC with UV detection at 350 nm using a Phenomenex Prodigy ODS(3) C18 column (5 μm, 250×4.6 mm) eluted with 40% acetonitrile/60% 60 mM triethylamine-phosphate buffer (v/v) pH 7.2. 2-(3-18F-Fluoro-4-methylamino-phenyl)-benzothiazol-6-ol had a retention time of 11 minutes at a flow rate of 2 mL/min (k′=5.5). The radiochemical purity was >99%, and the chemical purity was >90%. The radiochemical identity of 2-(3-18F-Fluoro-4-methylamino-phenyl)-benzothiazol-6-ol was confirmed by reverse phase radio-HPLC utilizing a quality control sample of the final radiochemical product co-injected with a authentic (cold) standard.
A cyclotron target containing 0.35 mL of 95% [0-18]-enriched water was irradiated with 11 MeV protons at 20 μA of beam current for 60 minutes, and the contents were transferred to a 5 mL reaction vial containing Kryptofix 222 (22.3 mg) and K2CO3 (7.9 mg) in acetonitrile (57 μL). The solution was evaporated to dryness three times at 110° C. under a stream of argon following the addition of 1 mL aliquots of acetonitrile. To the dried [F-18]fluoride was added 3 mg of 6-MOMO-BTA-N—Pr—Ots in 1 mL DMSO, and the reaction vial was sealed and heated to 85° C. for 30 minutes. To the reaction vial, 0.5 mL of MeOH/HCl (concentrated) (2/1 v/v) was added, and the vial was heated at 120° C. for 10 minutes. After heating, 0.3 mL of 2 M sodium acetate buffer was added to the reaction solution followed by purification by semi-prep HPLC using a Phenomenex Prodigy ODS-prep C18 column (10 μm 250×10 mm) eluted with 40% acetonitrile/60% 60 mM triethylamine-phosphate buffer (v/v) pH 7.2 at a flow rate of 5 mL/minute for 15 minutes, then the flow was increased to 8 mL/minute for the remainder of the separation. The product, [F-18]6-HO-BTA-N-PrF, eluted at 20 minutes in a volume of about 16 mL. The fraction containing [F-18]6-HO-BTA-N-PrF was diluted with 50 mL of water and eluted through a Waters C18 SepPak Plus cartridge. The SepPak cartridge was then washed with 10 mL of water, and the product was eluted using 1 mL of ethanol (absol.) into a sterile vial. The solution was diluted with 10 mL of sterile normal saline for intravenous injection into animals. The [F-18]6-HO-BTA-N-PrF product was obtained in 8±4% (n=8) radiochemical yield at the end of the 120 minute radiosynthesis (not decay corrected) with an average specific activity of 1500 Ci/mmol. The radiochemical and chemical purities of [F-18]6-HO-BTA-N-PrF were assessed by radio-HPLC with UV detection at 350 nm using a Phenomenex Prodigy ODS(3) C18 column (5 μm, 250×4.6 mm) eluted with 40% acetonitrile/60% 60 mM triethylamine-phosphate buffer (v/v) pH 7.2. [F-18]6-HO-BTA-N-PrF had a retention time of ˜12 minutes at a flow rate of 2 mL/minute (k′=6.1). The radiochemical purity was >99%, and the chemical purity was >90%. The radiochemical identity of [F-18]6-HO-BTA-N-PrF was confirmed by reverse phase radio-HPLC utilizing a quality control sample of the final radiochemical product co-injected with a authentic (cold) standard.
p-Anisidine (1.0 g, 8.1 mmol) was dissolved in anhydrous pyridine (15 ml), 4-nitrobenzoyl chloride (1.5 g, 8.1 mmol) was added. The reaction mixture was allowed to stand at room temperature for 16 hrs. The reaction mixture was poured into water and the precipitate was collected with filtrate under vacuum pressure and washed with 5% sodium bicarbonate (2×10 ml). The product was used in the next step without further purification. 1HNMR (30 MHz, DMSO-d6) δ: 10.46 (s, 1H, NH), 8.37 (d, J=5.5 Hz, 2H, H-3′,5′), 8.17 (d, J=6.3 Hz, 2H, H-2′,6′), 7.48 (d, J=6.6 Hz, 2H), 6.97 (d, J=6.5 Hz, 2H), 3.75 (s, 3H, MeO).
A mixture of 4-methoxy-4′-nitrothiobenzaniline (1.0 g, 3.7 mmol) and Lawesson's reagent (0.89 g, 2.2 mmol, 0.6 equiv.) in chlorobenzene (15 mL) was heated to reflux for 4 hrs. The solvent was evaporated and the residue was purified with flush column (hexane:ethyl acetate=4:1) to give 820 mg (77.4%) of the product as orange color solid. 1HNMR (300 MHz, DMSO-d6) δ: 8.29 (d, 2H, H-3′,5′), 8.00 (d, J=8.5 Hz, 2H, H-2′,6′), 7.76 (d, 2H), 7.03 (d, J=8.4 Hz, 2H), 3.808.37 (d, J=5.5 Hz, 2H, H-3′,5′), 8.17 (d, J=6.3 Hz, 2H, H-2′,6′), 7.48 (d, J=6.6 Hz, 2H), 6.97 (d, J=6.5 Hz, 2H), 3.75 (s, 3H, MeO). (s, 3H, MeO).
4-Methoxy-4′-nitrothiobenzanilides (0.5 g, 1.74 mmol) was wetted with a little ethanol (˜0.5 mL), and 30% aqueous sodium hydroxide solution (556 mg 13.9 mmol. 8 equiv.) was added. The mixture was diluted with water to provide a final solution/suspension of 10% aqueous sodium hydroxide. Aliquots of this mixture were added at 1 min intervals to a stirred solution of potassium ferricyanide (2.29 g, 6.9 mmol, 4 equiv.) in water (5 mL) at 80-90° C. The reaction mixture was heated for a further 0.5 h and then allowed to cool. The participate was collected by filtration under vacuum pressure and washed with water, purified with flush column (hexane:ethyl acetate=4:1) to give 130 mg (26%) of the product. 1HNMR (300 MHz, Acetone-d6) δ: 8.45 (m, 4H), 8.07 (d, J=8.5 Hz, 1H, H-4), 7.69 (s, 1H, H-7), 7.22 (d, J=9.0 Hz, 1H, H-5), 3.90 (s, 3H, MeO)
A mixture of the 6-methoxy-2-(4-nitrophenyl)benzothiazoles (22 mg, 0.077 mmol) and tin(II) chloride (132 mg, 0.45 mmol) in boiling ethanol was stirred under nitrogen for 4 hrs. Ethanol was evaporated and the residue was dissolved in ethyl acetate (10 mL), washed with 1 N sodium hydroxide (2 mL) and water (5 mL), and dried over MgSO4. Evaporation of the solvent gave 19 mg (97%) of the product as yellow solid.
To a solution of 2-(4′-aminophenyl)-6-methoxy benzothiazole (22 mg, 0.09 mmol) in glacial acetic acid (2.0 mL) was injected 1 M iodochloride solution in CH2Cl2 (0.10 mL, 0.10 mmol, 1.2 eq.) under N2 atmosphere. The reaction mixture was stirred at room temperature for 16 hr. The glacial acetic acid was removed under reduced pressure and the residue was dissolved in CH2Cl2. After neutralizing the solution with NaHCO3, the aqueous layer was separated and extracted with CH2Cl2. The organic layers were combined and dried over MgSO4. Following the evaporation of the solvent, the residue was purified by preparative TLC(Hexanes:ethyl acetate=6:1) to give 2-(4′-amino-3′-iodophenyl)-6-methoxy benzothiazole (25 mg, 76%) as brown solid. 1HNMR (300 MHz, CDCl3) δ (ppm): 8.35 (d, J=2.0 Hz, 1H), 7.87 (dd, J1=2.0 Hz, J2=9.0 Hz, 1H), 7.31 (d, J=2.2 Hz, 1H), 7.04 (dd, J1=2.2 Hz, J2=9.0 Hz, 1H), 6.76 (d, J=9.0 Hz, 1H), 3.87 (s, 3H).
To a solution of 2-(4′-Amino-3′-iodophenyl)-6-methoxy benzothiazole (5) (8.0 mg, 0.02 mmol) in CH2Cl2 (2.0 mL) was injected 1 M BBr3 solution in CH2Cl2 (0.20 ml, 0.20 mmol) under N2 atmosphere. The reaction mixture was stirred at room temperature for 18 hrs. After the reaction was quenched with water, the mixture was neutralized with NaHCO3. The aqueous layer was extracted with ethyl acetate (3×3 mL). The organic layers were combined and dried over MgSO4. The solvent was then evaporated under reduced pressure and the residue was purified by preparative TLC (Hexanes:ethyl acetate=7:3) to give 2-(3′-iodo-4′-aminophenyl)-6-hydroxybenzothiazole (4.5 mg, 58%) as a brown solid. 1HNMR (300 MHz, acetone-d6) δ (ppm): 8.69 (s, 1H), 8.34 (d, J=2.0 Hz, 1H), 7.77 (dd, J1=2.0 Hz, J2=8.4 Hz, 1H), 7.76 (d, J=8.8 Hz, 1H), 7.40 (d, J=2.4 Hz, 1H), 7.02 (dd, J1=2.5 Hz, J2=8.8 Hz, 1H), 6.94 (d, J=8.5 Hz, 1H), 5.47 (br., 2H). HRMS m/z 367.9483 (M+calcd for C13H9N2OSI 367.9480).
A mixture of 4-methylaminobenzoic acid (11.5 g, 76.2 mmol) and 5-methoxy-2-aminothiophenol (12.5, g, 80 mmol) was heated in PPA (˜30 g) to 170° C. under N2 atmosphere for 1.5 hr. The reaction mixture was then cooled to room temperature and poured into 10% K2CO3 solution. The precipitate was filtered under reduced pressure. The crude product was re-crystallized twice from acetone/water and THF/water followed by the treatment with active with carbon to give 4.6 g (21%) of 6-Methoxy-2-(4-methylaminophenyl)benzothiazole as a yellow solid. 1HNMR (300 MHz, acetone-d6) δ: 7.84 (d, J=8.7 Hz, 2H, H-2′ 6′), 7.78 (dd, J1=8.8 Hz, J2=1.3 Hz, 1H, H-4), 7.52 (d, J=2.4 Hz, 1H, H-7), 7.05 (dd, J1=8.8 Hz, J2=2.4 Hz, H-5), 6.70 (d, J=7.6 Hz, 2H, H-3′ 5′), 5.62 (s, 1H, NH), 3.88 (s, 3H, OCH3), 2.85 (d, J=6.2 Hz, 3H, NCH3)
To a solution of 2-(4′-Methylaminophenyl)-6-methoxy benzothiazole (20 mg, 0.074 mmol) dissolved in glacial acetic acid (2 mL) was added Ic1 (90 μL, 0.15 mmol, 1.2 eq, 1M in CH2Cl2) under N2. The reaction was allowed to stir at room temperature for 18 hr. The glacial acetic acid was then removed under reduced pressure. The residue was dissolved in CH2Cl2 and neutralized with NaHCO3. The aqueous layer was extracted with CH2Cl2 and the organic layers were combined, dried over MgSO4 and evaporated. The residue was purified with preparative TLC (Hexane: EA=2:1) to give 2-(4′-methylamino-3′-iodophenyl)-6-methoxy benzothiazole (8 mg, 27%) as brown solid. 1HNMR (300 MHz, CDCl3) δ (ppm): 8.39 (d, J=2.0 Hz, 1H), 7.88 (d, J=9.0 Hz, 1H), 7.33 (d, J=2.2 Hz, 1H), 7.06 (dd, J1=2.2 Hz, J2=9.0 Hz, 1H), 6.58 (d, J=9.0 Hz, 1H), 3.89 (s, 3H, OCH3).
To a solution of 2-(4′-methylamino-3′-iodophenyl)-6-methoxy benzothiazole (12 mg, 0.03 mmol) dissolved in CH2Cl2(4 mL) was added BBr3 (400 μl, 0.4 mmol, 1M in CH2Cl2) under N2. The reaction was allowed to stir at room temperature for 18 hr. Water was then added to quench the reaction and the solution was neutralized with NaHCO3, extracted with ethyl acetate (3×5 mL). The organic layers were combined, dried over MgSO4 and evaporated. The residue was purified with preparative TLC (Hexane: EA=7:3) to give 2-(4′-methylamino-3′-iodophenyl)-6-hydroxy benzothiazole (5 mg, 43%) as brown solid. 1HNMR (300 MHz, CDCl3) δ (ppm): 8.37 (d, H=2.0 Hz, 1H), 7.88 (dd, J1=2.0 Hz, J2=8.4 Hz, 1H), 7.83 (d, J=8.8 Hz, 1H), 7.28 (d, J=2.4 Hz, 1H), 6.96 (dd, J1=2.5 Hz, J2=8.8 Hz, 1H), 6.58 (d, J=8.5 Hz, 1H), 2.96 (s, 3H, CH3).
To a suspension of 2-(4′-nitrophenyl)-6-methoxy benzothiazole (400 mg, 1.5 mmol) in CH2Cl2 (10 mL) was added BBr3 (1M in CH2Cl2, 10 mL, 10 mmol). The reaction mixture was stirred at room temperature for 24 hr. The reaction was then quenched with water, and extracted with ethyl acetate (3×20 mL). The organic layers were combined and washed with water, dried over MgSO4, and evaporated. The residue was purified by flash chromatography (silica gel, hexanes:ethyl acetate=1:1) to give the product as a yellow solid (210 mg, 55%). 1HNMR (300 MHz, Acetone-d6) δ (ppm): 9.02 (s, OH), 8.41 (d, J=9.1 Hz, 1H), 8.33 (d, J=9.1 Hz, 1H), 7.96 (d, J=8.6 Hz, 1H), 7.53 (d, J=2.4 Hz, 1H), 7.15 (dd, J1=8.6 Hz, J2=2.4 Hz, 1H).
To a solution of 2-(4′-nitrophenyl)-6-hydroxy benzothiazole (50 mg, 0.18 mmol) dissolved in acetone (7 mL, anhydrous) was added K2CO3 (100 mg, 0.72 mmol, powdered) and MsCl (200 ul). After stirring for 2 hrs, the reaction mixture was filtered. The filtrate was concentrated and the residue was purified by flash column (silica gel, hexane:ethyl acetate=4:1) to give 2-(4-nitrophenyl)-6-methylsulfoxy benzothiazole (44 mg, 68%) as pale yellow solid. 1HNMR (300 MHz, acetone-d6) δ (ppm): 8.50-8.40 (m, 4H), 8.29 (d, J=2.3 Hz, 1H), 8.23 (d, J=8.9 Hz, 1H), 7.61 (dd, J1=2.3 Hz, J2=8.9 Hz, 1H).
To a solution of 2-(4′-nitrophenyl)-6-methylsulfoxy benzothiazole (35 mg, 0.10 mmol) dissolved in ethanol (10 mL) was added SnCl2.2H2O (50 mg). The reaction mixture was heated to reflux for 1.5 hr. The solvent was then removed under reduced pressure. The residue was dissolved in ethyl acetate (10 mL), washed with 1N NaOH, water, dried over MgSO4. Evaporation of the solvent afforded 2-(4′-aminophenyl)-6-methylsulfoxy benzothiazole (21 mg, 65%) as pale brown solid. 1HNMR (300 MHz, CDCl3) δ (ppm): 8.02 (d, J=6.2 Hz, 1H), 7.92 (d, J=8.7 Hz, 2H), 7.84 (d, J=2.4 Hz, 1H), 7.38 (dd, J1=2.4 Hz, J2=6.2 Hz, 1H), 6.78 (d, J=8.7 Hz, 2H), 2.21 (s, 3H, CH3).
To a solution of 2-(4′-methylaminophenyl)-6-hydroxy benzothiazole (300 mg, 1.17 mmol) dissolved in CH2Cl2 (20 mL) was added Et3N (2 mL) and trifluoroacetic acid (1.5 mL). The reaction mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (30 mL), washed with NaHCO3 solution. Brine, water, and dried over MgSO4. After evaporation of the solvent, the residue was dissolved in acetone (20 ml, pre-dried over K2CO3), K2CO3 (1.0 g, powered) was added followed by MsCl (400 mg, 3.49 mmol). The reaction mixture was stirred at room temperature and monitored with TLC until starting material disappeared. The residue was then filtrated. The filtrate was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (30 mL), washed with NaHCO3 solution. Brine, water, and dried over MgSO4. After evaporation of the solvent, the residue was dissolved in EtOH and NaBH4 was added. The reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated and the residue was dissolved in water, extracted with ethyl acetate (20 ml×3), the extracts were combined and dried over MgSO4. After evaporation of the solvent, the residue was purified with flash column (hexanes/ethyl acetate=8:1) to give the product (184 mg, 47.0%) as brown solid. 1HNMR (300 MHz, CDCl3) δ (ppm): 7.94 (d, J=8.8 Hz, 1H), 7.87 (d, J=8.7 Hz, 2H), 7.77 (d, J=2.3 Hz, 1H), 7.30 (dd, J1=8.8 Hz, J2=2.3 Hz, 1H), 6.63 (d, J=8.7 Hz, 2H), 3.16 (s, CH3), 2.89 (s, NCH3).
To a solution of 2-(4′-aminophenyl)-6-methanesulfonoxy benzothiazole or 2-(4′-methylaminophenyl)-6-methylsulfoxy benzothiazole (1 mg) in 250 μL acetic acid in a sealed vial was added 40 μL of chloramines T solution (28 mg dissolved in 500 μL acetic acid) followed by 27 μL (ca. 5 mCi) of sodium [125I]iodide (specific activity 2,175 Ci/mmol). The reaction mixture was stirred at r.t. for 2.5 hrs and quenched with saturated sodium hydrogensulfite solution. After dilution with 20 ml of water, the reaction mixture was loaded onto C8 Plus SepPak and eluted with 2 ml methanol. For deprotection of the methanesulfonyl group, 0.5 ml of 1 M NaOH was added to the eluted solution of radioiodinated intermediate. The mixture was heated at 50° C. for 2 hours. After being quenched by 500 μL of 1 M acetic acid, the reaction mixture was diluted with 40 mL of water and loaded onto a C8 Plus SepPak. The radioiodinated product, having a radioactivity of ca. 3 mCi, was eluted off the SepPak with 2 mL of methanol. The solution was condensed by a nitrogen stream to 300 μL and the crude product was purified by HPLC on a Phenomenex ODS column (MeCN/TEA buffer, 35:65, pH 7.5, flow rate 0.5 mL/min up to 4 min, 1.0 mL/min at 4-6 min, and 2.0 mL/min after 6 min, retention time 23.6). The collected fractions were loaded onto a C8 Plus SepPak. Elution with 1 mL of ethanol gave ca. 1 mCi of the final radioiodinated product.
A. Study Participant Information
A total of 21 [11C]PIB PET studies have been performed on 16 subjects. Five of the 21 studies were test/re-test studies. Table 1 lists subject characteristics including age, mini-mental state examination (MMSE) score and gender. Three of these subjects are from a large famial AD (FAD) kindred (highlighted in grey in Table 1; M+ indicates mutation carrier, S+ indicates symptomatic dementia). Subjects were recruited and evaluated, receiving their diagnosis in a consensus conference of experienced neurologists, psychiatrists, neuropsychologists and clinicians according to published criteria. (Lopez et al., Neurology 55:1854-1862, 2000).
Of the five control subjects, C-4 was a young control age-matched to the M+S−FAD subject and C-5 was a M−S-sibling of the AD-5 M+S+FAD patient. Of the 5 MCI subjects, MCI-2 and MCI-5 have been cognitively stable while the others have had slow, mild but progressive cognitive decline limited only to memory at the time of the [11C]PIB study.
All subjects underwent fully-dynamic [11C]PIB (90 min) and simplified FDG (25 min) PET imaging studies. The procedure was well-tolerated and completed by all subjects. All five subjects who were asked to return for a re-test within 21 days agreed and again completed the study without problems. The [11C]PIB studies were conducted over 90 min after slow (20 sec) bolus injection of [11C]PIB. PET scanning was performed using a Siemens/CTI HR+ scanner (see section C.2.3.2).
B. Blood Collection Data for Determining [11C]PIB Radioactivity Concentration in Plasma
Arterial blood sampling was successfully performed in all 21 studies. The average [11C]PIB radioactivity concentration in plasma is shown, in
C. ROI-Based SUV Analyses
Examples of [11C]PIB time-activity curves in terms of SUV data are shown in
D. Analysis of Amyloid Imaging Data for Distinguishing Control, AD and MCI Patients
The preliminary [11C]PIB data from the 5 controls, 5 AD and 5 MCI subjects and the one M+S−FAD subject studied to-date were analyzed using several methods over 60 and 90 min of data (all subjects scanned for 90 min). The analyses included 5- and 4-parameter, 3-compartmental models (including vascular volume) and the Logan graphical method with arterial blood data as the input function (Logan-ART) and with cerebellar data as the input function (Logan-CER). Also, a Patlak analysis and a 2-compartment model were applied but neither described the data as well as the 3-compartment or Logan methods based upon goodness-of-fit criteria and regression coefficients. Therefore, the Logan method was employed in the analyses discussed below. The Logan distribution volume ratios (DVRs) shown in
Examples of the initial results obtained from the first 16 subjects using the Logan graphical method (using of arterial blood or cerebellar data as input functions) are shown in
The data shown in
1) The present pharmacokinetic analysis is consistent with the SUV analysis presented above. That is, the AD subjects clearly show higher DVR values than the control subjects in the cortical areas known to have heavy amyloid deposition, such as frontal and posterior cingulate cortex. Furthermore, AD patients and controls are equivalent in areas without neuritic plaques such as mesial temporal cortex and cerebellum; and
2) The Logan-CER-60 method produces results that are very similar to the 60 and 90 min arterial blood input methods. Although the DVR values determined with cerebellum as input and using only the first 60 min of data (Logan-CER-60) are systematically lower, the correlation with the 90 min arterial input data (Logan-ART-90) is very good (R2=0.989;
The Logan-CER-60 DVR data for all 5 controls, 5 MCI, 5 AD subjects and the one M+S−eFAD subject are shown in
E. Voxel-Based Analyses
[11C]PIB SUV images show marked [11C]PIB retention in association cortices and little retention in cerebellum (
These image data were not corrected for cerebral atrophy but still demonstrate greater [11C]PIB localization in cortical areas for the AD subject (
F. Re-Test Reliability
Both the Logan-ART-90 min method and the Logan-CER-60 min method proved to be very stable in a test/re-test study. The Logan-ART-90 method showed a mean test/re-test variation of 8.5±5.3% over all areas studied and the Logan-CER-60 method showed an even better test/re-test variation of 5.1±4.5% (
This section describes efforts to extend the above-summarized, quantitative PIB studies, with nine additional subjects (n=24), to include an evaluation of simplified methodology, i.e., methods that do not require arterial blood sampling, for PIB PET imaging studies. In these examples, the performance of several methodological simplifications for PIB PET were compared to that of the fully-quantitative method-of-choice, Logan graphical analysis, based on arterial input and 90 minutes of emission data. (Logan-ART-90 min will be referred to as ART90 in this section.) The simplifications included a shorter scan duration, the use of image-derived cerebellar or carotid time-activity data, in lieu of an arterial input function, and a single-scan method based upon the ratio of standardized uptake values (SUV) in the region-of-interest normalized to the cerebellar SUV. These examples illustrate a PIB PET methodology that can be simply and reliably applied across the AD disease spectrum, while providing a good compromise between accuracy and precision in the PIB retention measures.
A. Human Subjects
PIB PET imaging was performed for 24 subjects, which included healthy controls (3M, 5F: 65±16 years), and subjects with a diagnosis of either MCI (8M, 2F: 72±9 yrs) or AD (6M: 67±10 yrs). Table 2 below describes the subject characteristics including age, MMSE score and gender. The procedure was well tolerated by all subjects.
†Eight subjects underwent a second “re-test” PIB PET study within 28 days of the “test” or baseline study. The time interval between scans is shown in parentheses
‡Individual ages are listed in parentheses. Ages are not listed in the order of the subject ID to preserve anonymity.
B. Imaging
High specific activity (SA) PIB PET studies were performed in the 8 healthy controls (dose: 488.4±107.3 MBq; SA: 47.8±21.7 GBq/μmol), 10 MCI patients (dose: 510.6±77.7 MBq; SA: 45.9±24.9 GBq/μmol), and 6 AD patients (dose: 514.3±96.2 MBq; SA: 31.3±18.1 GBq/μmol). Average regional CSF factors are shown in Table 3 below. The regional CSF correction factors, which were determined from each individual subject's SPGR MR data, showed no significant differences for any group comparison, using the one-sided non-parametric Wilcoxon rank test after FDR correction.
C. Input Function Comparisons
The input functions determined via hand-drawn arterial samples were compared to those derived by carotid VOI placement. Metabolite-corrected input functions determined by arterial sampling and carotid VOI placement were corrected for injected dose and body mass (% ID*kg/g) to allow population-average input functions (n=24) to be generated for the purpose of comparison (
D. Data Analysis
This section includes a description of the basic PET data, a summary of the primary results that were observed across all methods, method-specific performance issues, and evaluations of method performance. Comparisons of the mean PIB retention measures focused on differences between AD and control subject groups because PIB retention for the ten MCI subjects was found to range across control and AD levels. That is, MCI subjects do not represent a homogeneous group distinct from either controls or AD subjects.
Tissue Data. Tissue:cerebellar radioactivity concentration ratios were computed for each brain region. In posterior cingulate, the region that showed the highest degree of PIB retention in AD subjects, the VOI:CER ratios reached a plateau at a value of approximately 2.5:1 after 45 min while control subjects maintained ratios of approximately 1:1 for all primary amyloid-binding areas (
Overall Results. Table 4 below lists the mean values measured in AD and control subjects, for each method, across the 11 regions. All methods yielded significantly higher DVR or SUVR values for AD subjects compared to controls in regions known to contain amyloid in AD. The most significant differences (p<0.001, see statistical methods) were generally observed in PCG, ACG, FRC, PAR, LTC, CAU (Table 4). Lesser differences (0.001≦p≦0.05) were observed for OCC, SMC, and MTC. There were no significant differences in PIB retention between AD and control subjects in regions that are known to be virtually free of amyloid pathology in mild-to-moderate AD subjects, such as SWM and PON (p>0.20). No method yielded significant group differences in the cerebellar DV or SUV value for AD patients relative to controls (p>0.25).
†p < 0.05, AD < controls, 1-sided
‡p < 0.001, AD < controls, 1-sided
Three of the MCI subjects (M-2, 5, 9) showed patterns of PIB retention that were indistinguishable from the control group. Five MCI subjects (M-1, 3, 4, 7, 8) demonstrated patterns of retention that were characteristic of the AD subject group. Two MCI subjects (M-6, 10) tended to be higher than controls in PCG or FRC (
Standardized uptake value (SUV). The single (summed) scan tissue ratios that were computed over either 40-60 min (SUVR60) or 40-90 min (SUVR90) were found to be in agreement for both the AD and control subject groups. In controls, the regional SUVR60 ratios ranged from 1.11±0.13 (CAU) to 1.80±0.13 (PON), while the SUVR90 tissue ratios ranged from 1.14±0.13 (CAU) to 1.76±0.14 (PON). In AD subjects, the regional SUVR60 and SUVR90 values ranged from 1.38±0.19 (MTC) to 2.80±0.28 (PCG) and from 1.40±0.20 (MTC) to 2.88±0.30 (PCG), respectively.
Logan Graphical Analyses. The Logan graphical analysis generally provided estimates of DV (arterial or carotid input) and DVR (cerebellar input) values with high regression correlations (r2>0.97) in 10 of 11 regions. These results are consistent with the data satisfying the linearity condition required by the Logan analysis. For the SWM, correlations were generally lower (0.7<r2<0.99) than for other regions, particularly when the dataset was truncated to 60 min.
Parametric images of DVR measures obtained using the ART90 and CER90 analyses show similar patterns and levels of PIB retention (
Multilinear Regression. The multilinear regression analysis (MA1) was applied using a reference tissue input in an exploratory manner for a high binding and low binding region (PCG and MTC) over 90 min. The MAI DVR estimates in these regions were essentially identical to those determined using CER90. This suggests that noise-induced bias is not a factor at the VOI level for the determination of the PIB Logan DVR. As a result of this excellent agreement, the remainder of the examples focus solely on Logan analysis results.
Simplified Reference Tissue Analysis. The use of SRTM with only 60 min of data resulted in highly variable outcome measures, spuriously overestimated values, and deviations in regional rank order. For this reason, typical SRTM results were obtained using 90 min of data. SRTM90 detected significant differences (p<0.001) in DVR values between control and AD subjects in several cortical and subcortical regions (Table 4). For the 90 min data set, average RI values in control subjects ranged from 0.40±0.20 (SWM) to 0.99±0.15 (OCC). RI values in AD subjects were comparable to controls in most regions, ranging from 0.35±0.08 (SWM) to 0.97±0.09 (OCC). In both AD and control subjects, only MTC and SWM showed RI values consistently lower than 0.75. The most notable group difference in average RI values was evident for PAR (controls: 0.86±0.06 and AD: 0.74±0.08), while PCG was more similar (controls: 0.91±0.06 and AD: 0.85±0.10). The aforementioned RI values were not corrected for partial volume effects.
E. Evaluation Criteria
Rank Order. The regional rank order of outcome measures averaged for the six AD subjects was well conserved across all nine simplified methods as each identified PCG as the region with the greatest PIB retention, followed by ACG and other cortical regions, including PAR, FRC, and LTC (Table 5).
PIB binding in caudate exceeded that of SMC, OCC, and MTC. White-matter-containing regions, such as PON and SWM, were among the lowest in terms of regional rank order in AD subjects. In control subjects, white-matter containing regions such as PON and SWM occupied the highest ranks.
The individual subject rank order was well-maintained across methods and regions. In general, CAR90 showed the best agreement with ART90 in terms of individual subject rank order (
Methods that involved the truncation of the dataset to 60 min (ART60, CER60, CAR60, SUVR60) identified other subjects, A-4 or A-2, as the AD subject with the greatest PIB retention rather than A-1. Among the control subjects, the ART90 DVR values indicate subjects C-1 and C-6 to have elevated levels of PIB retention relative to other controls in PCG, while subjects C1-1 and C1-2 appear to have elevated PIB retention in FRC (
Test-Retest Variability. The intra-subject, or test-retest, variability of the simplified PIB retention measures was evaluated for the eight subjects retested within 28 days of the initial PIB PET scan, using the percent difference and ICC measures (see Statistical methods). Table 6 summarizes the variability measures and shows that favorable margins of test-retest variability were observed that were generally within ±10% across methods and regions, except for SWM (6.0-23.8%).
7.5
4.9
3.6
4.6
5.2
6.9
8.8
5.9
5.9
5.0
7.9
9.2
4.0
5.6
2.9
3.0
2.7
4.6
3.5
5.9
2.4
3.0
4.4
4.4
8.2
5.9
4.0
5.5
5.5
7.1
12.3
11.0
7.9
8.2
12.8
12.9
4.4
5.9
4.0
4.2
5.8
6.2
4.0
6.9
3.6
4.2
3.3
5.0
4.3
8.0
4.1
4.3
4.9
5.3
†Primary areas of interest are shown in boldface.
For most regions, the CER60 and CER90 methods showed lowest test-retest variability with averages within ±4.4% and ±4.6% respectively. Interestingly, the cerebellar-based SRTM method showed somewhat greater variation than either CER60 or CER90, averaging ±6.2% across all regions. The SUV-based methods were reproducible as well, averaging ±5.3% and ±5.0% across regions for SUVR60 and SUVR90, respectively. The greatest test-retest variability (within 10%) was observed for the arterial based methods. Greater variability was observed with a shorter scan duration, as is the case for CAR60 (+12.9%) and ART60 (9.2%), while that for the 90 min measures was less. ART90 and CAR90 performed similarly well, with test-retest variability across the 11 regions averaging ±6.9% and ±7.1%, respectively.
Bias and Correlation: Bias in the PIB retention measures was examined over low-DVR (ART90 PCG DVR<1.8, n=13) and high-DVR (ART90 PCG DVR>1.8, n=11) groups (see
Across all subjects (n=24), the PCG and FRC DVR values determined using each simplified method were highly correlated (r2=0.921-0.995) with the ART90 DVR values (
Effect Size: The effect size measure reflects the level of variation of a given measure across subjects (inter-subject variability) and separation of the group mean PIB retention values. It was often noted that arterial-based methods tended to be more variable than cerebellar-based methods and the 60 min data tended to be more variable than the 90 min data. For the controls, CER60 was generally associated with the least variation in DVR across subjects that was less than 10% for all regions except ACG (14%) and FRC (16%). ART60, CAR60, and SRTM90 yielded CV % values that were greater than 10% for 9 of 11 regions (excluding cerebellum) (Table 3). For the AD group, greater DVR coefficients of variation were most often observed for ART90 and ART60 ranging from about 10-20% in primary areas-of-interest.
All methods consistently separated control and AD groups and resulted in large Cohen's effect sizes for regions with high PIB retention. The greatest Cohen's effect sizes (d) were observed in the PCG and ranged from about 6.9 (SUV methods) to 4.6 (SRTM90). The magnitude of the effect sizes reflects that clear separation of mean PIB retention values is achieved between control and AD subjects. Table 7 lists the range of effect sizes in PCG, FRC, MTC, and PON.
The PON region is not expected to differ between AD and control subjects, and thus has an effect size that varies about zero. Significant group differences in PIB retention were detected between AD and control groups for all regions except SWM and PON.
F. Discussion
In the discussion that follows, four levels of simplification will be examined: 1) shortening the scan period from 90 to 60 min; 2) substituting an arterial input function derived from a volume-of-interest defined over the carotid artery for arterial plasma-based input (CAR60/90); 3) replacing arterial input completely with an image-driven analysis method, such as the non-invasive Logan analysis (CER60/90) and SRTM90; and 4) use of a late single-scan measure of the radioactivity distribution (SUVR60/90). Within each level of simplification, performance compared to the benchmark quantitative method, ART90, was assessed by four criteria: a) fidelity of regional rank order; b) test-retest variability; c) % bias and correlation; and d) Cohen's effect size. It is acknowledged that the ART90 method is a “relative” benchmark, as there are currently no post-mortem measures of the true amyloid deposition in these subjects against which different measures of PIB retention can be independently compared.
With the exception of SRTM60 noted below, all other methods of simplification maintained regional rank order very well. Therefore, rank order will not be discussed individually in the sections below. Test-retest variability relates to the ability to detect small changes over time in amyloid deposition (in natural history studies) or amyloid clearance (in anti-amyloid therapy trials). Methodologic bias in this study was defined as the difference in outcome measures of a simplified method to the ART90 outcome measure, normalized to the ART90 value. Effect size is an indication of the ability of a method to detect small but statistically significant differences in amyloid deposition between groups.
Shortened Scan Interval:
The first level of simplification examined the possibility of acquiring the PIB PET scan for a shorter period of time, 60 min rather than 90 min. In general, analysis methods that used 90 min of emission data performed somewhat better, although methods that used 60 min of emission data yielded useful data as judged by the evaluation criteria employed. The most notable exception was the application of SRTM using 60 min of data, which resulted in spurious values, high intersubject variability, and aberrations of regional rank order. A shorter scan duration was associated with substantially higher test-retest variability in the case of ART60 and CAR60 (Table 6), although for ART60 this measure was still within the +10% margin generally considered acceptable for most PET radiotracers (Smith, G. S. et al. Synapse, 1998; 30(4): 380-92; Volkow, N. D. et al. J Nucl Med, 1991; 34: 609-13). This is of greatest concern for longitudinal studies that require reliable repeat measures of PIB retention. Truncation to 60 min did not result in a significant change in the level of methodologic bias for CAR60 or SUVR60, but CER60 showed a larger negative % bias compared to CER90 (
Carotid VOI-Derived Arterial Input Function:
The next level of simplification sought to obviate arterial line placement in favor of an input function derived from a volume-of-interest defined over the carotid artery on the early frames of the reconstructed PIB image. While this method is limited in that it does not provide an estimate of the unchanged fraction of PIB in plasma on an individual basis, the use of a population average metabolite correction represented a satisfactory substitute for individual data. Of all methods examined, the 90 min carotid-based method (CAR90) provided PIB DVR estimates which most closely reflected ART90 DVR values and were the least biased relative to ART90 for both low- and high-DVR subjects (
Reference Tissue-Based Input Function:
A further simplification is realized when estimates of the arterial input function are obviated in favor of a completely image-driven analysis method, such as the non-invasive Logan analysis (CER60, CER90) and SRTM, which rely on the identification of a consistent tissue region devoid of radiotracer specific binding, such as the cerebellum (Logan, J. et al. J Cereb Blood Flow Metab, 1996; 16(5): 834-40; Lammertsma, A. A. et al. Neuroimage, 1996; 4(3 Pt 1): 153-8.). The CER60 and CER90 methods resulted in DVR estimates that were negatively biased with respect to ART90 DVR measures (
The non-invasive Logan methods (CER60 and CER90) had the lowest test-retest variability of any method examined, averaging +4.4% and +4.6% across all regions, respectively. SRTM90 showed slightly higher test-retest variability (+6.2% across regions) than CER60 or CER90, though this level of variability would be considered to represent a satisfactory level of performance for a PET imaging agent. Inter-subject variability in the control group was substantially higher for SRTM90 than either CER60 or CER90, though in the AD group the methods were more comparable. This fact largely explains the larger effect sizes observed for CER60 and CER90 compared to SRTM90.
Late Single Scan Measure:
The greatest degree of simplification is realized using a method based on a late single-scan measure of the static radioactivity distribution, such as the SUV-based methods (SUVR90 and SUVR60). These assessments do not require the collection of a complete dynamic emission dataset or arterial input function data. Rather, they are based solely on regional differences in the distribution of radioactivity in the brain over some later time interval following radiotracer injection, after which specific binding of radiotracer is expected to be a major component of brain radioactivity concentrations. Because of its simplicity, the SUV measure is frequently employed in clinical studies where it can be impractical to employ quantitative analysis methods that require dynamic imaging or input function determination. To eliminate a major source of variability in the determination of SUV, the time interval for the evaluation of the SUV parameter must be chosen such that the change in the SUV value over the interval is relatively small in comparison to the SUV value itself (Beaulieu, S. et al. J Nucl Med, 2003; 44(7): 1044-50).
In the case of in vivo PET studies, the SUVR reflects the relative contributions of specific and non-specific binding to the measured signal and is therefore more comparable to the DVR value, which has been corrected for non-specific binding by normalizing regional DV estimates with the cerebellar DV value. For the PIB data, the ratio of tissue (amyloid containing) to cerebellar radioactivity was relatively constant beyond 40 min post-injection in both AD an control subjects (
Selecting a Method-of-Choice:
The selection of a method-of-choice will depend upon the nature of the particular application. All of the simplified methods examined in this study provided results that compared well to the ART90 method and overall the similarities were greater than the differences between methods. Nevertheless, each method has certain advantages and disadvantages for specific purposes.
Scan Duration:
In general, it appears that all methods that use 90 min of data consistently outperform the corresponding method using only the first 60 min. Acquisition of this data requires a full 90 min dynamic scan for CER90, CAR90, SRTM90, but all of the data necessary for the SUVR90 analysis can be obtained by having the subject in the scanner during just the 40-90 min time window. The comparable performance of the SUVR60 method using the 40-60 min window suggests that it may be possible to optimize/shorten the 40-90 min window even further without loss of performance. This may be especially important for the study of severe AD patients who may not be able to tolerate a full 90 min of emission data acquisition. In addition to the shorter scan time, other advantages of the SUVR method include simplicity of application (making it more applicable to routine clinical studies), superior PCG effect size (6.9), very good test-retest reproducibility (5.0%) and a large dynamic range (evidenced by a positive bias vs. ART90). A disadvantage shared by the SUVR, CER and SRTM methods is greater influence of any inaccuracies contributed by the cerebellar data used as reference. This would be particularly apparent if there was detectable amyloid deposition in the cerebellum.
Cross-Sectional Inter-Group Comparisons:
In the primary areas-of-interest (e.g. PCG, FRC, PAR), all methods demonstrated the ability to distinguish AD and control subjects without any overlap between groups (
Studies that Correlate Amyloid Load with Other Variables:
For some purposes, it may be important to distinguish subjects across a large spectrum of amyloid deposition and perhaps correlate amyloid deposition with other variables (e.g., neuropsychological measures, regional FDG or MRI measures, blood or CSF measures of amyloid). A biased but reliable method could provide DVR values that are restricted in dynamic range or erroneously distributed depending on the uniformity of bias. Thus statistical correlation of the PIB retention measures with other indices could be limited when the degree of bias is not uniform across the range of expected values, as is the case with the CER60 and CER90 methods. An additional difficulty that could arise in relating other variables to measures of amyloid deposition is the lack of normally distributed data, especially when all subject groups are combined. Since most measures of correlation, most notably Pearson's and Spearman's, are based on the assumption of bivariate normal distributions, correlations would only be accurate within subgroups which appear to be normally distributed. This is perhaps of paramount concern for the study of MCI subjects, which is a heterogeneous group of subjects that spans the entire range of PIB retention, but has a distinctly bimodal distribution. The study of MCI subjects is perhaps one of the more interesting and promising applications of PIB, as there are no effective non-invasive indicators of progression of amyloid pathology. In this situation, it would be advantageous to apply the simplified method with the lowest and most uniform bias (e.g., CAR90) although at the expense of higher test-retest variability.
Longitudinal Studies:
A third type of comparison study is one in which longitudinal examinations of PIB retention are made in the same subject to study the natural history of disease progression or the response to anti-amyloid therapies. In this instance, it is desirable to have the most reliable repeat measure possible in order to be sensitive to what could potentially be small changes in the degree of amyloid deposition or resorption between serial examinations. This may be an important consideration when planning a longitudinal study using PIB where one would expect the differences in PIB retention between serial examinations to be small, or a study which focused on MCI or normal aging where there would be the expectation of a lower specific binding signal. While the cerebellar methods CER90 and CER60 have shown the lowest test-retest variability, one must again consider whether or not this advantage is offset by the inherent bias in these methods. However, the low test-retest variability makes CER90 an attractive method for detecting small effects of experimental anti-amyloid therapies over time, particularly in cases with low levels of amyloid deposition that must ultimately be the principle target of these therapies.
In summary, when it is not possible or desirable to obtain arterial-based input data, several simplified methods can be valid alternatives to quantitative arterial-based analyses. The SUVR90 method may be the method of choice when simplicity of calculations and short in-scanner time are the overriding concerns. The CAR90 method may be the method of choice when comparison across a large range of amyloid deposition and minimization of cerebellum-derived artifacts is the prime concern. The CER90 method may be the method of choice for natural history studies and treatment trials, particularly in subjects with lower levels of amyloid deposition, when the detection of small interval changes is paramount. SUVR90 may perform better in treatment trials in subjects at the high end of amyloid deposition. In practice, the data necessary for all of these analyses will be available after a 90-minute dynamic PIB scan, and so the decision regarding method of choice does not necessarily need to be made beforehand.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
As used herein and in the following claims, singular articles such as “a”, “an”, and “one” are intended to refer to singular or plural.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/23618 | 7/1/2005 | WO | 00 | 10/4/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60584495 | Jul 2004 | US |
