Technologies

Targeted Therapy Targeting Apoptosis Targeting Cardiotoxicity Targeting Bacteria


Targeted Therapeutics for Cancer

The use of cytotoxic drugs for cancer therapy has spawned the development of targeted drug delivery in attempts to reduce toxicity to normal tissues, sparing life-threatening side effects and improving therapy. Apoptosis and necrosis are two distinct pathways of cell death. However, both cell death processes converge on one critical biomarker, that is, externalization of phosphatidylserine (PS). PS is abundant in the tumor microenvironment, possibly due to the stress conditions, such as hypoxia, acidity, thrombin, inflammatory cytokines, and reactive oxygen species. Our strategy takes advantage of the bulk expression of PS at the tumor site as an apparent biomarker for the spatial- and temporal-controlled delivery of therapeutics to treat cancer. Since many disorders and pathological conditions, such as cancer, are the result of deregulation of cell death, PS has become a key target for the delivery of therapeutics. Agents that can selectively target PS on membrane surfaces and distinguish them from the near-neutral membrane surfaces of normal human cells have promising potential as imaging probes, drug delivery agents, and targeted molecular therapeutics

The lead product candidate consists of Small Molecule Drug Conjugate Cancer (SMDC) containing the potent anti-cancer agent SN-38, a topoisomerase I inhibitor, with a proprietary novel linker group. The linker group is designed to provide stability in plasma but be susceptible to enzymatic cleavage in the tumor microenvironment. Characterization of this product candidate included successful demonstration of retention of PS binding affinity, plasma stability, cellular toxicity to colon and gastric cancer cells (nontoxic to normal cells), and a relatively high tolerated dose in mice. Of particular significance was the ability of the compound to inhibit tumor growth in a mouse colon cancer xenograft model. Our product candidate was compared directly to Irinotecan, a SN-38 prodrug, with both drugs dosed intravenously at 40 mg/kg, twice weekly for two weeks. Equivalent drug weight dosing results in the SMDC drug conjugate containing only 40% of the Irinotecan dose. Results showed distinct advantage in the drug conjugate-dosed mice compared to Irinotecan-dosed mice at 22 days post treatment initiation. The tumors in the drug conjugate-dosed mice did not appear to grow beyond the size at initiation of treatment. Additionally, our SMDC-T1 was shown to have an “amplification” effect with the initial cytotoxicity causing elevated levels of PS which in turn increases recruitment of the SMDC-T1 conjugate to the tumor site, further enhancing its therapeutic effect.

 





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Targeting Apoptosis and Necrosis with Imaging Agents:

Zn-DPA is a small molecule Annexin-V mimic with the ability to selectively target anionic phospholipid residues exposed on the membranes of apoptotic and necrotic cells, cancer cells, and endothelial cells of tumor vasculature. The Zn-DPA molecule is currently being developed for molecular imaging applications such as near-infrared (NIR) optical imaging and nuclear imaging (PET/SPECT). A direct application of this molecule involves imaging of tumor response to chemotherapy, as this often involves one or more modes of cell death such as apoptosis and necrosis. Early assessment of therapy at the molecular level would provide a important tool for preclinical screening cancer therapeutics and allow a change of treatment when therapy is ineffective, thereby limiting unnecessary treatment and associated costs.

Size Matters
(A) Monitoring tumor response to chemotherapy: Palmowski et. al. (European Radiology 2014, 24:363-370) reported the increase in apoptosis during anti-angiogenic therapy was significantly better detected with the low molecular weight Zn-DPA probe PSVue794 than with AnnexinVivo750.
Conclusions:

(i) The reason for the higher accumulation of PSVue794 is most probably related to its 20 times smaller size, which enables better delivery of the probe to the target cells. Moreover, the smaller size is likely to enable a higher loading degree of targeting ligands per membrane surface area as compared to Annexin V, which reaches saturation at approximately 50 phosphatidylserine molecules per cell.

(ii) PSVue provides an advantage over Annexin V in the preclinical selection of new anti-angiogenic therapeutic molecules.

(B) Targeting traumatic brain injury: Smith et. al. (ACS Neuroscience Chemical Science 2012, 3:530-537) demonstrated the ability of a near-infrared fluorescently labeled Zn-DPA probe, named PSS-794, to detect cell death in a brain cryolesion mouse model that replicates certain features of traumatic brain injury.

Representative in vivo near-infrared fluorescence montages of PSS-794, Tracer-794 (control dye), and Annexin-Vivo 750 accumulation in a brain cryoinjury mouse model. A precooled metal cylinder was applied to the head of each mouse for 60 s followed by intravenous injection of either PSVue (PSS-794) (3.0 mg/kg), Tracer-794 (3.0 mg/kg), or Annexin-Vivo 750. Images were acquired at the indicated time points after probe injection. N=5


In vivo quantification of PSvue (PSS-794), Tracer-794, and Annexin-Vivo 750 accumulating in a 60 s brain cryoinjury mouse model. Target to nontarget ratios (T/NT) were calculated by region of interest (ROI) analysis of the digital images. Shapes were drawn around the site of the cryoinjury (target, T) and around an equivalent site on the lower back (nontarget, NT) and the mean pixel intensities (MPI) were recorded. T/NT ± SEM (N = 5).



Conclusions:

(i) The in vivo epifluorescence imaging showed that the maximum T/NT ratio with PSVue 794 (6.77 ± 0.47) occurred at about 3 h after probe injection and was about 2 times higher than the maximum T/NT ratio with Annexin Vivo-750.

(ii) PSVue 794 can be used to visualize cell death in an adapted cryolesion mouse model of TBI.







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Monitoring Cardiotoxicity and Assessing the Effectiveness of Cancer Therapy

Duramycin is a natural antibiotic with 19-amino acidsproduced by Streptoverticillium cinnamoneus . Duramycin recognizes apoptotic and necrotic cells by binding to (PE) with high affinity and specificity (Zhao et. al. J. Nuclear Medicine 2008, 49(8):1345-1352). The overall structure of Duramycin assumes a compact cyclic configuration, with a single binding pocket that specifically interacts with PE. Stabilized by 3 internal thioether linkages, Duramycin is the smallest known polypeptide that has a defined 3-dimensional binding site..

Attributes of 99mTc-Duramycin as an Imaging Agent

  • 99mTc-Duramycin retains its PE binding activity after radiolabeling it's highly stable in vivo.
  • Binds apoptotic and necrotic cells with high affinity.
  • Rapid blood clearance with half-life of ~2-4 minutes.
  • Rapid renal clearance and low hepatic retention.
  • Ischemic/necrotic myocardium can be detected within 10-20 minutes post iv injection in animal AMI models.
  • Little washout from infarcted myocardium over time.
  • Infarcted tissue is detectable at least 48 hours post infarction but no uptake in older infarcts.
  • No toxicity expected with the current dosage.

99mTc-Duramycin has been studied successfully in animal models for the imaging of myocardial infarct, stroke, atherosclerotic plaque, cardiotoxicity and cancer. Recently exquisite SPECT/CT images from a rat model of cardiac ischemia/reperfusion were published showing excellent focal infarct uptake and coupled with low systemic background and rapid renal/urinary clearance (Audi et al./ Nuclear Medicine and Biology 39 (2012), 821-825.)

MTTI has licensed from the Medical College of Wisconsin rights to an allowed US patent covering the synthesis and characterization of 99mTc-HYNIC-Duramycin for the non-invasive imaging of phosphatidylethanolamine (PE) residues as well as HYNIC modification of Duramycin and its radiolabelling. Additionally, two other CIPs are pending, which include PET imaging with F-18 and Ga-68 labeled Duramycin.

Whole-body and cardiac imaging in rat model of myocardial ischemia/reperfusion

Studies from a rat model of cardiac ischemia/reperfusion have produced exquisite SPECT/CT images showing excellent focal infarct uptake coupled with low systemic background and rapid renal/urinary clearance.

Representative SPECT/CT images of 99mTc-Duramycin uptake in ischemically damaged cardiac tissue in vivo. The CT, SPECT and SPECT/CT fusion images are shown in the left, middle and right columns, respectively. Radioactivity uptake in the left ventricular free wall is highlighted with an arrow in the tomographic images.

We envision that Tc-99m Duramycin will be useful for the following:

Monitoring cardiotoxicity:

A clinically important therapy-limiting toxicity is myocardial damage secondary to Doxorubicin and radiation therapies is paramount. Chemotherapy, radiotherapy, and other cancer treatments are known to trigger tumor cell apoptosis, an event in which PS and PE are exposed on the dying tumor cells and their microvesicles. We believe that the detection by 99mTc-Duramycin imaging of the newly exposed PE resulting from the therapy-related apoptosis would provide information regarding whether a patient is benefiting or not from the treatment. Importantly, because of its sensitivity, quantifiability, and clinical translatability, a radiotracer imaging-based procedure for detecting and localizing apoptosis holds unique promise.

Assessment of Effectiveness of Cancer Therapy:

It is of significant interest to be able to identify early in the course of treatment those patients who are benefiting from chemotherapy or other forms of cancer therapy. Establishing predictive markers of chemoresponse would help to individualize therapy and improve survival of cancer patients. A sensitive, specific molecular imaging readout of cancer therapeutic efficacy, just after beginning a new regimen, would accelerate and simplify disease management.

To date, both the decline of tumor markers and the assessment of morphologic changes in tumor size by radiological methods such as CT or MRI have been used for assessing the tumor response during treatment. However, the decline of serum tumor markers depends on certain metabolic pathways, which may vary from patient to patient and the marker response during chemotherapy may be misleading. Radiological imaging for the assessment of tumor response may also be of limited value due to the delay between the start of treatment and tumor shrinkage as well as the inability of radiological imaging methods to differentiate vital tumor from necrosis or scar tissue in case of residual lesions. Thus, additional means for the monitoring of treatment response would be helpful to identify non-responding patients and to avoid ineffective and toxic treatment.

Preliminary studies by our collaborators (Elvas, Wyffels et. a. J. Nucl Med 2014, 55, 1240) in Belgium have demonstrated the ability of 99mTc-Duramycin to detect the increase in externalized PE on tumor cells after radiotherapy and chemotherapy. These preliminary data support the proof of concept for the use of 99mTc-Duramycin to monitor response to cancer therapy.


 




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Targeting Bacteria with Imaging Agents

Smith, et. al. (Chem. Comm.,2008, 20, 2331-2333; Bioconjugate Chemistry, 2010, 21, 1297-1304) demonstrated that fluorescently-labeled Zn-DPA (PSVue 794) can target anionic phospholipids within the bacterial envelopes of Gram-positive and Gram-negative bacterial cells, such as Staphylococcus aureus and Escherichia coli, versus the relatively uncharged plasma membranes of normal human cells. In vivo studies involving intravenous injection of this probe into living mice led to fluorescence localization at the sites of bacterial infection, and demonstrated that the probe can differentiate bacterial infection from sterile inflammation.

(A) Distinguish bacterial infection from inflammation

Example 1. S. pyrogene bacteria- Liu et. al. (J. Nuclear Medicine Communications 2012, 39, 709-714) showed In-111 labeled PSVue biotin targeted S. pyogenes-infected mice similarly to PSVue 794 . The significantly higher accumulation in the live bacterial infection thigh compared to that of the LPS-induced inflammation thigh suggests that Zn-DPA may be a promising imaging agent to distinguish between bacterial infections and sterile inflammations.

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Example 2. Mouse model with S. aureus bacteria: White et. al. (Bioconjugate Chemistry, 2010, 21, 1297-1304) demonstrated Zn-DPA (PSVue) selectively accumulated at bacterial infection sites in the thigh muscles of nude mice. PSVue can be used to optically image bacterial infection while not responding to sterile inflammation in living mice.

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