DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group II in the reply filed on 11/10/2025 is acknowledged. Claims are pending, of which claims are withdrawn from consideration at this time as being directed to a non-elected invention. Claims 14-23 encompass the elected invention and are examined herein on the merits for patentability.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 14-18 are rejected under 35 U.S.C. 103 as being unpatentable over Tafoya et al. (J Mag. Res. Imaging, 2017, 46(2), p. 574-588) in view of Bok et al. (Biomed. Optics Express, 2015, 6(9), p. 3303-3312).
Tafoya teaches superparamagnetic nanoparticle-enhanced MRI of Alzheimer’s disease plaques and activated microglia in 3X transgenic mouse brains. Given the attractive properties of SPIONs, our emphasis has been to develop targeted MRI-active, nontoxic super-paramagnetic nanoparticles slightly larger than natural viruses that penetrate the BBB and bind to pathological sites within the brain. Optimization of the MRI sequences for the detection of both amyloid-targeted SPIONs and novel, microglial-targeted, iron/platinum (FePt) micellar nanoparticles are taught. Our two secondary goals were to use the optimized MRI sequences to confirm our earlier findings in PS1/APP2X Tg mice 8 that our amyloid-targeted SPIONs were useful for measuring for Ab plaque density and its reduction due to treatment with resveratrol, and to show that our novel Iba-1-targeted FePt nanoparticles (SM80s) could be used to measure microglial activation in the 3X Tg mouse brain and the reduction in neuroinflammation due to treatment with resveratrol (page 575).
Synthesis and conjugation of FePt stealth micelles is taught on page 576. FePt nanoparticles were added to DSPE, DSPE-PEG, DSPE-PEG-biotin and LissRho. Anti-Iba-1 antibodies were strepavidinated using the Lightning-Link SA kit according to the manufacturers protocol and were added to SIPP micelles and mixed overnight at room temperature. Free antibody was removed… washing with distilled water, and eluting off the magnetic separator with PBS containing 1% Polysorbate to make the finished SM80 (SIPP/micelle/PS80) nanoparticles.
Mice were injected with the nanoparticle preparations,
Ex Vivo MRI at 11.7T is shown on page 576. Feed control mice, and two remaining resveratrol-treated mice were injected with either of the two nanoparticle preparations by tail vein and then killed 24hours later. The brains of these mice were perfused in situ with phosphate-buffered formalin (10%) by cardiac puncture, quickly removed, and fixed in 4% phosphate-buffered formalin. The four selected brains used for ex vivo MRI were stored in this formalin solution at 48C until prepared for use.
Plaques and sites of activated microglia appeared in the images as localized hypointense regions 10–300 pixels in area.
Antibody-conjugated superparamagnetic nanoparticles penetrated the BBB and bound to the 50 µm diameter Ab plaques, and the microglia surrounding these lesions, producing regions of large local susceptibility gradients that dephased the tissue water proton magnetization and gave rise to areas of MR signal loss (Fig.1A).
Resveratrol treatment reduced neuroinflammation MRI of the brains from SM80-injected mice revealed areas of neuroinflammation as hypointense regions surrounding the amyloid plaques (Fig. 7I). More than 1700 sites (Table1) of neuroinflammation were found in the untreated control group of 3X Tg mice (Fig. 3H). Chronic treatment of these mice with 100 ppm resveratrol in their diet reduced this number of microglia by a factor of 4 to 450 (Figs.3H, 7K,L; Table 1) a value consistent with our previous optical microscopy findings. 9 The Z-score once again was observed 9 to increase on resveratrol treatment (Fig. 3G) where we found that the most probable Z was 27.9 in the control mice, which increased to 32.7 in the resveratrol-treated animals (P < 0.0001). We also observed that the use of a RARE sequence, with its refocusing of the transverse dephasing, showed about a factor of 10 fewer sites of neuro-inflammation (Fig. 7F) than were found using a susceptibility-weighted sequence.
As far as neuroinflammation was concerned, application of the susceptibility-weighted FLASH sequence to control, untreated mice injected withanti-Iba-1-conjugated SM80s revealed large numbers (Table1) of microglia (Figs. 7I,J), which markedly decreased on treatment with resveratrol (Figs. 7K,L) (page 582).
Resveratrol treatment lowered the number of activated microglia in the brain. It was shown that resveratrol treatment (Fig. 8F) lowered the number of neuroinflammatory sites in these brains by 4-fold. Comparison of both the number of Ab and neuroinflammatory sites detected with MRI and their mean Z-scores showed that the FePt SM80s performed with similar efficacy to the SPIONs used (page 584).
In this study, we showed that two innovative MRI superparamagnetic contrast agents (anti-amyloid-SPIONs and anti-Iba1-SM80s) that penetrated the BBB aided the MRI detection of AD plaques and activated microglia in a 3X trans-genic mouse model of AD by increasing their Z-score (contrast-to-noise ratio). This mouse model reproduces many of the pathological features that are observed in AD in humans, including amyloid plaque deposition, tauopathy, neuroinflammation, BBB compromise and neuronal death (page 585).
Our study also showed that MRI, aided by targeted superparamagnetic nanoparticles, constituted a noninvasive method for measuring the efficacy of drug treatment using resveratrol, a phytochemical being studied for the treatment of AD to reduce both amyloidosis and neuroinflammation (page 585).
Our novel FePt nanoparticles were successfully used to detect active microglia using much the same MR approach as with Ab detection by the SPIONs (page 586).
Tafoya does not teach the detection of microglia as imaging, diagnosing or monitoring treatment of stroke.
Bok teaches that microglia are resident macrophages in the brain, and typically exist in the resting state characterized by ramified morphology. In response to brain injuries such as ischemic stroke, they are rapidly activated undergoing morphological changes to amoeboid morphology and displaying several activation markers including CD68 and major histocompatibility complex (MHC) class II. Activated microglia exert various immune functions including phagocytosis of dead cells and production of many cytokines, reactive oxygen, and nitrogen species. Although it is not yet clear whether these processes mediated by activated microglia are detrimental or beneficial in ischemic stroke, chronically activated microglia have been reported to exacerbate neurological injury leading to the development of depressive-like behavior. Given their critical roles in contributing neuroinflammation thereby exacerbating the progression of various brain diseases including stroke, Alzheimer’s disease, and Parkinson’s disease, activated microglia have recently been exploited as an attractive candidate for non-invasive imaging to monitor neuroinflammation. Studies have reported temporal dynamics of neuroinflammation in mice and humans by PET imaging for 18 kDa translocator protein (TSPO), the outer mitochondrial membrane cholesterol transporter largely expressed in activated microglia. While these studies have demonstrated a direct clinical application for PET imaging of activated microglia, it is still difficult to visualize them at the cellular and the molecular levels. Here we report in vivo imaging of activated microglia by establishing intracranial window chamber (ICW) in a mouse model of focal cerebral ischemia by using two-photon microscopy (TPM). Since its first demonstration in 1990, TPM has been applied to many in vivo studies of various organs including the brain with its advantageous features of higher imaging depths with minimal phototoxicity compared to other 3D microscopy techniques. In this report, we administered fluorescent labeled antibodies to wild-type mice subjected for focal cerebral ischemia and found that significant levels of Iba-1 and CD68 positive activated microglia were observed in the ipsilateral compared to the contralateral side of the infarct and that indomethacin significantly lowered CD68 signals in TPM imaging. We believe that in vivo imaging of ICW coupled with TPM would be a useful tool to better understand cellular and molecular processes involved in neuroinflammation (page 3303).
Injection of PerCP-Iba-1 antibodies in GFP mice revealed a strong Iba-1 signal in ICW of mice undergone MCAO but not in sham animals by TPM [Fig. 1(d)], likely to be due to the intact blood brain barrier in the latter. A number of those Iba-1-positive microglia observed in ICW of MCAO mice were in the ramified morphology, consistent with the immunostaining results [Fig. 1(b)], indicating that they were microglia (page 3308).
We then injected CD68 antibodies together with Iba-1 antibodies in MCAO mice bearing ICW in order to detect activated microglia by TPM. In this experiment, we set up ICW either in the ipsilateral or the contralateral side of the infarct [Fig. 2(a)]. We observed that Iba-1 and CD68 signals were much stronger in the ipsilateral side than the contralateral side of the infarct (page 3309).
To determine whether ICW imaging by TPM can be utilized to monitor the therapeutic efficacy of agent(s) lowering microglial activation, we tested indomethacin (Indo), a nonsteroidal anti-inflammatory drug, which has been previously reported to dampen microglial activation in a rat model of focal cerebral ischemia (page 3310).
It would have been obvious to one of ordinary skill in the art at the time of the invention to extend the teaching of Iba-t targeted superparamagnetic micellar nanoparticle-enhanced MRI of Alzheimer’s disease plaques and activated microglia for detection, imaging and monitoring therapy of stroke/ischemia when the teaching of Tafoya is taken in view of Bok. One would have been motivated to do so, with a reasonable expectation of success, because Bok teaches that, in addition to Alzheimer’s disease, microglia are associated with stroke/ischemia, and labeled Iba-1 allows for non-invasive imaging to monitor neuroinflammation in mice subjected for focal cerebral ischemia. Further, each of Tafoya and Bok teach imaging as means of monitoring therapeutic treatment after administration of Iba-1 labeled imaging agents, such as monitoring therapeutic efficacy of agent(s) lowering microglial activation.
Claim(s) 14-23 are rejected under 35 U.S.C. 103 as being unpatentable over Tafoya et al. (J Mag. Res. Imaging, 2017, 46(2), p. 574-588) in view of Bok et al. (Biomed. Optics Express, 2015, 6(9), p. 3303-3312, in further view of Jiang et al. (Stroke, 2010, 41, p. 410-414.)
The rejection over Tafoya in view of Bok is applied as above.
With regard to claims 19-23, Tafoya and Bok do not specifically teach the time period for performing MRI after treatment to determine the effect of treatment.
Jiang teaches that MRI is a vital tool for the measurement of acute stroke and has been used to visualize changes in activation patterns during stroke recovery. There is emerging interest on using MRI to monitor the structural substrates of spontaneous recovery and neurorestorative treatment of stroke. The use of MRI and its associated challenges to measure vascular and neuronal remodeling in response to spontaneous and therapy induced stroke recovery is described.
Experimental studies suggest that cell-based or pharmacological-based neurorestorative treatments can enhance brain reorganization and substantially improve functional recovery when treatment is initiated up to weeks after stroke. Neurorestorative treatments amplify endogenous processes of brain plasticity, including angiogenesis and neuronal remodeling through neurogenesis and axonal reorganization, which likely contribute to improvement in neurological function after stroke. Current understanding of angiogenesis, neuronal remodeling after stroke, however, derives mainly from regional histological measurements, which do not allow dynamic assessment of tissue remodeling. In contrast, MRI can noninvasively monitor the temporal profiles of functional recovery and tissue remodeling after stroke (page 410).
Newly formed cerebral vessels are inherently leaky, because it can take several weeks to form a functional blood– brain barrier. An early approach in detecting angiogenesis by MRI was to monitor changes in blood volume with time, which may reflect the growth of new blood vessels (page 411).
Enhanced angiogenesis colocalized with increases of cerebral blood flow and CBV at 6 weeks after treatment and colocalized with transient increases of blood-to-brain transfer constant (Ki) of Gd-DTPA with a peak at 2 to 3 weeks after cell therapy (page 411).
It would have been obvious to one of ordinary skill in the art at the time of the invention to performing MRI after treatment to determine the effect of treatment of stroke over a period of weeks when the teachings of Tafoya and Bok are taken in view of Jiang. While Tafoya and Bok address monitoring the therapeutic efficacy of agent(s) lowering microglial activation, a time period of monitoring is not specifically recited. However, one would have been motivated to perform monitoring by MRI over a period of weeks because Jiang teaches that one can noninvasively monitor the temporal profiles of functional recovery and tissue remodeling after stroke, and that it can take several weeks to form a functional blood-brain barrier, such as performing imaging at 2, 3 and 6 weeks for example. Accordingly, one of ordinary skill would have found it routine to monitor effect of therapeutic treatment by MRI using contrast over a period of week(s) after administering treatment.
Conclusion
No claims are allowed at this time.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to LEAH H SCHLIENTZ whose telephone number is (571)272-9928. The examiner can normally be reached Monday-Friday, 8:30am - 12:30pm EST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, MICHAEL HARTLEY can be reached at 571-272-0616. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/LHS/
/Michael G. Hartley/Supervisory Patent Examiner, Art Unit 1618