ABSTRACT
An injectable nanoemulsion formulation has been developed, by Cav-Con Inc., which is especially useful for highly selective delivery of incorporated (lipophilic) dyes, labels, or drugs to various types of solid tumors and certain other lesions. All these lesions consistently display an increased (cell-surface) expression and/or activity of "lipoprotein receptors", including notably the (class B) scavenger receptor referred to as SR-BI [cf. recent book: D'Arrigo, J.S. (2011) Stable Nanoemulsions: Self-Assembly in Nature and Nanomedicine, 436 pp., Elsevier Science, Amsterdam and Oxford]. Such data on SR-BI expression and function are noteworthy; namely, SR-BI has emerged as the lipoprotein receptor primarily involved in the enhanced endocytosis (i.e., enhanced intracellular uptake) of "lipid-coated microbubbles (LCM) / nanoparticle-derived" nanoemulsions into hyperproliferative-disease sites [see D'Arrigo (2011) reference above]. Hence, for such lesions including localized brain-injury sites [cf. below], the overexpression of (cell-surface) SR-BI facilitates targeted imaging and targeted drug-delivery therapy, via the above nanoemulsion [ e.g., Filmix® ], of those types of disease or injury -- including traumatic brain injury. This proposed R&D program is likely to demonstrate (in Phase 1) an efficacious "actively targeted" agent for improved identifying and monitoring of mild brain injuries, using MRI, as well as for their selective treatment.
BACKGROUND AND RATIONALE
The corporation's (injectable) nanoemulsion formulations, which are especially useful for "actively targeted" imaging and/or chemotherapy, comprise several key lipid components that can be adjusted for specific applications. [For a detailed review of the relevant scientific and patent literature, associated know-how, advantages over competing technologies, target markets, etc., see D'Arrigo (2011) reference above.] Briefly, the above 2011 book reviews and analyzes much experimental (in vivo) data which collectively demonstrate that this type of stable lipid nanoemulsion, upon intravenous injection, is capable of "active targeting" of various lipophilic molecules (imaging agents and/or drugs) to hyperproliferative-disease sites -- which commonly overexpress certain cell-surface receptors, including the (class B, type I) scavenger receptor (known as "SR-BI").
An example of the above-mentioned [cf. Abstract] "other" (noncancerous) disease/injury sites (involving proliferative processes), which overexpress scavenger receptors, concerns central-nervous-system (CNS) injury - that is, brain injury and/or spinal cord injury. Various published studies indicate increased scavenger receptor expression on "proliferating macrophages" and "activated astrocytes" arising after CNS injury [D'Arrigo (2011) reference above]. In this regard, the findings of Kureshi et al. [Neurosurgery 44:1047-1053 (1999)] have direct relevance to Filmix-related lipid nanoemulsions, that is, LCM (cf. Abstract) and particularly "dispersed LMN" (see below). As reviewed earlier (in greater detail in Sect. 14.2.2.2 of D'Arrigo (2011) reference above), these investigators reported that LCM [and/or agglomerations of the far more numerous and smaller "dispersed LMN" (undecipherable at that time) ] in Filmix agent , injected intravenously, displayed a readily measurable affinity to injured rat spinal cord.
More important (to the present proposal) is another application of LCM (and/or the "dispersed LMN [ lipid-mesophase nanoparticles ]" ) to CNS injury; namely, Ho et al. [Brain Res. Bull. 43:543-549 (1997)] studied the affinity of LCM to the site of a localized (thermal) brain injury. As reviewed previously (in more detail in Sect. 14.2.2.2 of D'Arrigo (2011) reference above), these authors point out it had been well documented earlier that in response to injury in the CNS, astrocytes are activated ; this process is accompanied by an increased content of GFAP, hypertrophy, and hyperplasia [Bignami & Dahl, Neuropath. Appl. Neurobiol. 2:99-110 (1976); Mathewson & Berry, Brain Res. 327:61-69 (1985); Miyake et al., Brain Res. 590:300-302 (1992); Nieto-Sampedro et al., Brain Res. 343:320-328 (1985); Schiffer et al., Brain Res. 374:110-118 (1986)] -- a process referred to as gliosis [Nieto-Sampedro et al., Brain Res. 343:320-328 (1985)]. In particular, Ho et al. observed that the influx of LCM began at the time when GFAP-positive cells began to appear, and it seemed likely that the LCM are initially attracted to the "reactive" astrocytes [ Ho et al., Brain Res. Bull. 43:543-549 (1997)].
In follow-up to these findings with brain injury, the experiment program with LCM (and/or dispersed LMN) was expanded to next examine the use of LCM to deliver 7ß-hydroxycholesterol (7ß-OHC) to a radiofrequency (thermal) lesion in the rat brain [Wakefield et al., Neurosurgery 42:592-598 (1998)]. ( 7ß-OHC and other oxysterols have been reported, by other groups, to inhibit astrogliosis as well as tumor cell proliferation both in vitro and in vivo [see D'Arrigo (2011) book above for listing of cross-references] ). The data obtained in this follow-up study indicate that both the number of activated astrocytes and the intensity of the GFAP-staining were reduced when treated with 7ß-OHC delivered by the LCM, while not affected by the same dose of intravenously injected 7ß-OHC in saline [Wakefield et al., Neurosurgery 42:592-598 (1998)]. It appears that the mechanism of this enhanced delivery of 7ß-OHC to the brain-injury site, by LCM (and/or dispersed LMN), shares common features with the "receptor (i.e., SR-BI)-mediated endocytic pathway" mechanism described earlier for the case of tumor cells [cf. Sect. 24.3 of D'Arrigo (2011) reference above]. This interpretation of the data receives additional indirect support from published findings, of other investigators, which document the expression of particularly SR-BI on astrocytes and vascular smooth muscle cells in adult mouse and human brains (as well as in Alzheimer's disease brain) [Husemann & Silverstein, Am. J. Pathol. 158:825-832 (2001)].
Therefore, the ultimate objective of "targeted imaging and/or chemotherapy" of neuro-injury sites, utilizing "active targeting" behavior of an intravenous agent, is particularly well-suited to the Filmix nanoemulsion (imaging-agent and chemotherapy) delivery vehicle -- since SR-BI has emerged as the lipoprotein receptor primarily involved in ligand-receptor binding of this lipid nanoemulsion vehicle at the relevant target cells [D'Arrigo (2011) reference above].
PRELIMINARY EVIDENCE
Yu et al. describe traumatic brain injury (TBI) as one of the most acute degenerative pathologies in the central nervous system, where in vivo indices enabling an assessment of TBI on a mechanistic basis have yet to be established. Their work in a rat model of TBI has been aimed at clarifying the aggressive and protective roles of glial responses to injury when combined with emerging anti-inflammatory and immunomodulatory treatments [Yu et al., J. Neurotrauma 27:1463-1475 (2010)]. Similarly, Batchelor et al. assert that inflammation in the CNS predominantly involves microglia and macrophages, and is believed to be a significant cause of secondary injury following trauma [Batchelor et al., J. Neurotrauma 25:1217-1225 (2008)]. This belief is consistent with earlier work in mice where macrophage scavenger receptor expression on resident microglia and recruited macrophages was detected 24 hours after brain injury (from cytotoxic agents) [ Bell et al., J. Neurocytol. 23:605-613 (1994) ] , and with later work by Nagamoto-Combs et al. demonstrating that "microglia/macrophage undergo prolonged activation" after TBI in the non-human primate brain. These latter authors further point out that impaired fine motor functions after TBI in humans and non-human primates often continue to improve months after injury, suggesting possible involvement of microglia and/or macrophages in the long-term recovery processes [Nagamoto et al., J. Neurotrauma 24:1719-1742 (2007)]. In view of all the foregoing considerations, it becomes evident that inflammation of brain tissue in the absence of infection (sterile inflammation) contributes to acute brain injury and chronic disease. Accordingly, Savage et al. have recently studied the inflammatory responses of glial cells in the presence of a relevant endogenous priming stimulus; interestingly, these authors report the acute-phase-protein serum amyloid A (SAA) [see below] acted as a sterile, endogenous, priming stimulus on glial cells [ Front. Immunol. 3: (published online 9-18-2012)].
With regard to the above recent finding, serum amyloid A (SAA) is a liver-derived "high-density lipoprotein (HDL)"-associated apolipoprotein, whose level in the blood increases up to 1,000-fold in response to various injuries including trauma (e.g., CNS injury), inflammation (e.g., human vascular plaques and Alzheimer's lesions), etc. Like other acute-phase reactants, the liver is the major site of SAA expression; however, SAA is also expressed in cells at inflammation sites, e.g., macrophage cell lines and within human atherosclerotic lesions [e.g., D'Arrigo (2011) reference above]. Baranova et al. point out that the importance of SAA in various physiological and pathological processes has generated considerable interest in the identity of the cell-surface receptor(s) that bind, internalize, and mediate SAA-induced proinflammatory effects; furthermore, these authors assert that the results of their study demonstrate that CLA-1 ( the human SR-BI ortholog [Vishnyakova et al., J. Biol. Chem. 278:22771-22780 (2003)] ) functions as an endocytic SAA receptor, and is involved in SAA-mediated cell signaling events associated with the immune-related and inflammatory effects of SAA [Baranova et al., J. Biol. Chem. 280:8031-8040 (2005)]. In addition, CLA-1 and SR-BI are highly expressed on monocytes/macrophages, cells known to be the primary sites of SAA uptake [Baranova et al., J. Biol. Chem. 280:8031-8040 (2005); Pearson et al., Curr. Opin. Immunol. 8:20-28 (1996)].
Lastly, Vishnyakova et al. point out that despite the fact that CLA-1 has not been studied as extensively as rodent SR-BI, the physiological role of (human) CLA-1 is generally assumed to be similar to that of rodent SR-BI [J. Biol. Chem. 278:22771-22780 (2003)]. Accordingly, the planned in vivo experiments, to be conducted in the second half of this proposed Phase 1 ("diagnosis and prognosis") project, will be carried out using a rodent neuro-injury model. ( These planned MRI experiments (see below) involve measuring changes in "bulk magnetic susceptibility" [cf. Magn. Reson. Med. 52:445-452 (2004); Neuroimage 46:658-664 (2009); Proc. 10th Annu. Mttg. SMRM, San Francisco, p. 1020, (1991)].)
TECHNICAL MATURITY
Briefly, the Filmix nanoemulsion is constructed entirely of nonionic lipids -- i.e., saturated glycerides, cholesterol, and cholesterol esters. In addition, this lipid composition (of the Filmix colloidal system, i.e., glycerides and cholesterol compounds) is similar to lipids contained in several types of plasma lipoproteins. Accordingly, when these Filmix colloidal particles (i.e., "lipid-coated microbubbles" [LCM] and related lipid nanoparticles) are injected into the bloodstream, they acquire (bind) plasma apolipoprotein(s). These bound apolipoprotein(s) are evidently recognized by the corresponding lipoprotein receptors, often found overexpressed on the surface membrane of hyperproliferative cells (e.g., tumor cells); specifically, confocal laser microscopy clearly demonstrates successful tumor-selective endocytosis of the Filmix lipid particles [Barbarese et al., J. Neuro-Oncology 26:25-34 (1995)]. The measured lipid nanoemulsion particle uptake by target cells (tumor cells) displays both temperature dependence and energy dependence. Moreover, this Filmix nanoemulsion delivery vehicle is physically capable of carrying high-contrast in vivo imaging agents ( for example, lipophilic dyes [or other small lipophilic molecules for other in vivo imaging modalities] ) with these nanoemulsion particles into the target cells [cf. D'Arrigo (2011) reference above].
The safety of Filmix nanoemulsion has been tested in animals, in compliance with the Principles of Good Laboratory Practices (GLP). Specifically, the Filmix product vials (lot no. 12-29-89) were tested at an independent GLP contractor, Leberco Testing Inc., and passed USP sterility and LAL pyrogen tests. Thereafter, this lot of Filmix nanoemulsion product was employed in two acute intravenous (i.v.) toxicity studies in rabbits and dogs at another independent GLP contractor, Pharmakon Research International Inc. No significant signs of gross toxicity or mortality were observed at a dosage of 4.8 mL/kg, which is several-fold higher than those doses employed in preclinical (ultrasound-imaging) efficacy studies in animals. (Similarly, efficacy testing of Filmix nanoemulsion at Colorado State University in 53 dogs, at a dose of 0.3 mL/kg, has never resulted in any post-injection adverse aftereffects in these diverse dogs.) Besides the above acute studies, subchronic toxicological studies were also conducted with Filmix agent in rats and rabbits [see www.netplex.net/~cavcon for full description of details].
The in vivo experiments on rats, to be conducted (under formal subcontract) in the 2nd half of this proposed Phase 1 project, will involve improvements in current MRI brain scanning technology. Specifically, the "actively targeted" Filmix® nanoemulsion will be employed (via i.v. injection), in these MRI experiments on rats, to reveal brain sites of increased endocytosis (i.e., mirroring increased "SAA-uptake receptor" activity [see "Preliminary Evidence" sect. above] ) and, thereby, enhance the identification of abnormal brain structure and/or function. Accordingly, this lipid-nanoemulsion agent's "active targeting" behavior [see "Background and Rationale" sect. above] has the likely capacity to act as a novel biomarker of brain injury -- including neuroinflammation and traumatic brain-injury.
Note that this same "Filmix lipid nanoemulsion agent" ( alone, i.e., with no added paramagnetic [or other imaging] label ) was utilized successfully in an earlier, preliminary, published MRI report [ Huang et al., Proc. 12th Annu. Mttg. Soc. Magnetic Resonance in Med., p. 757, (1993) ] to provide focally enhanced detection of brain tumors in rats via MRI. The documented ability of LCM to concentrate rapidly and selectively in tumor tissue [see D'Arrigo (2011) reference above], with no accumulation in surrounding normal tissue, has been found to cause a significant change in the "bulk magnetic susceptibility" (BMS) inside tumors in vivo as detected by MRI (see below). Since the BMS of air is about 0.4 ppm (37 C) and that of tissue or blood is about 9 ppm [Albert et al.,NMR Biomed. 6:7 (1993)], the susceptibility difference created by the accumulation of LCM in the tumor region was expected to generate local magnetic field gradients that shorten the average T2 and/or T2* values of the water proton spins inside the tumor. Therefore, the affected region should appear darker in a T2- or T2*-weighted MR image, with the larger contrast effect in the latter [ Huang et al., Proc. 12th Annu. Mttg. Soc. Magnetic Resonance in Med., p. 757, (1993)].
MRI contrast enhancement by the Filmix nanoemulsion agent (which contains "lipid-coated microbubbles" [ LCM ] and related lipid nanoparticles) was examined in a rat brain-tumor model (9L gliosarcoma) and the time courses of the enhancement were recorded. The MRI experiments were performed on a clinical 1.5 T GE SIGNA Advantage Unit with the combination of a body transmitter coil and a homemade 4-cm diameter solenoid receiver coil that was placed around the rat's head. The rat's body axis was perpendicular to the magnetic field. A three-dimensional (3D) volume data set was acquired using a 3D GRASS (Gradient Recalled Acquisition in Steady State) sequence in about 8 min, with a 256 x 256 data matrix size, 8 cm FOV, 5º flip angle, TE = 15 ms, and TR = 60 ms. A total of 28 contiguous slices with 0.7 mm thickness were collected. Prior to MRI examination, the tumor-bearing rat was anesthetized (i.p.) and the tail vein was catheterized with a 27-gauge lymphangiogram needle. A dose of the LCM agent (Filmix®, 0.3-1.4 ml/kg) was delivered by intravenous injection through the tail vein over 1-2 min. One 3D volume GRASS data set was acquired before, and another at about 3-5 min after, the injection of LCM agent. Rats were imaged 7, 14, and 19 days after the tumor cell implantation. A dose (0.5 mmol/kg) of a standard clinical MRI contrast agent (Magnevist [Berlex]) stock solution (500 mM GdDTPA2-) was later delivered through the tail vein of the rat with a 19-day tumor to also define the tumor region in a T1-weighted image using a spoiled-GRASS sequence [ Huang et al., Proc. 12th Annu. Mttg. Soc. Magnetic Resonance in Med., p. 757, (1993) ]. The pre-LCM (i.e., pre-Filmix®) GRASS images showed no delineation of the 7- and 14-day tumors. The post-LCM T2*-weighted GRASS images showed one or several dark focal regions (area ranging from 0.11 to 1.29 mm2) at the location of the tumor, but not elsewhere in the normal tissues within the field of view [cf. D'Arrigo (2011) reference above].
The older the tumor, the more abundant and larger the focal regions. The T1-weighted post-Magnevist image of the rat with a 19-day tumor exhibited a much larger (~ 23 mm2) bright region in the brain that is presumably the area of pathologically altered blood-brain barrier. All the dark regions in the T2*-weighted post-LCM image were located within the Magnevist-enhanced area [ Huang et al., Proc. 12th Annu. Mttg. Soc. Magnetic Resonance in Med., p.757, (1993)]. Since the volume fraction of the total LCM subpopulation in the Filmix® nanoemulsion is roughly 2.2 ~ 2.3 x 10-6, the BMS of the Filmix® stock solution is almost the same as that of pure saline solution, about 9 ppm [Albert et al., NMR Biomed. 6:7 (1993)]. Thus, the unconcentrated stock solution would not cause BMS-based contrast in tissue. These results suggest that the LCM are indeed concentrated directly and uniquely in the tumor, with no accumulation in normal brain tissue [ Huang et al., Proc. 12th Annu. Mttg. Soc. Magnetic Resonance in Med., p. 757, (1993)]. This is probably due, in part, to pathological alterations in the intrinsic tumor capillaries [e.g., Ward et al., Cancer 34:1982-1991 (1974); Stewart et al., Acta Neuropathol. 67:96-102 (1985) ] as well as to receptor-mediated endocytosis [cf. D'Arrigo (2011) ref. above], which appears to significantly increase the local volume fraction of the microbubbles and therefore change the local BMS value. The change of the BMS in the tumor region finally results in contrast enhancement in T2- and T2*-weighted images. [ For comparison, the most common MRI contrast agent approved for use (> two decades) clinically for brain tumor enhancement, GdDTPA2-, shortens both the 1H2O T1 and T2 relaxation times by hyperfine mechanisms; whereas, the Filmix nanoemulsion agent (alone, i.e., with no added "paramagnetic metal" or other label) is expected to shorten T2 and/or T2* by bulk magnetic susceptibility (BMS) mechanisms. More generally, while MRI and ultrasonography are both widely available medical imaging techniques and are free of ionizing radiation, preoperative diagnosis and localization of brain tumors is possible noninvasively only with the former since MRI is not limited by the presence of bone (an intact skull) [e.g., D'Arrigo, Drug News & Perspec. 4:164-167 (1991)]. ] To conclude regarding the above-described MRI experiments on tumor-bearing rats, the regions appearing very dark on the GRASS images must have the most concentrated LCM inside the tumor, and possibly inside tumor cells themselves [ Huang et al., Proc. 12th Annu. Mttg. Soc. Magnetic Resonance in Med., p. 757 (1993); cf. D'Arrigo (2011) ref. above].
By the end of 1-2 years of further development if selected, this proposed R&D program is likely to demonstrate "proof of concept", in animals, toward an efficacious "actively targeted" (i.v.) Filmix nanoemulsion agent [containing no "paramagnetic metal" ions or complexes]. The agent may well both: 1) improve the identification and analysis (via the LCM subpopulation in Filmix®) of brain injuries using MRI; and 2) accurately detect changes longitudinally to monitor, and potentially also provide targeted chemotherapy (via mostly the accompanying lipid-nanoparticle subpopulation in Filmix®) to assist, the patient's recovery and/or prognosis.
PROPOSAL TEAM (PRINCIPAL INVESTIGATOR) EXPERIENCE, AND INTELLECTUAL PROPERTIES
[ For details concerning Dr. J.S. D'Arrigo's past patents (with links to full text) and current intellectual property, peer-reviewed journal
publications (with links to abstracts and sample figures), books (with Table of Contents, preface, and all chapter summaries), grants, and previous
development activities, please see www.netplex.net/~cavcon. ] As a brief background, Dr. D'Arrigo received a B.A. (in Chemistry) from Queens College,
SUNY in NYC, NY; attended Univ. of Wisconsin Medical School for two years and passed (Part I of) National Medical Boards; and then received a Ph.D.
degree (in Neuroscience) at the Brain Research Institute, UCLA. Following an N.I.H. postdoctoral fellowship, he held faculty positions
(in neurophysiology) at the Univ. of Utah College of Medicine and the Univ. of Hawaii School of Medicine for a total of 12 years, and was a
visiting fellow (in colloids / interfacial chem.) at the Inst. of Advanced Studies, Australian National Univ., Canberra, Australia (1 year) -- before
entering industry (in biotechnology). Dr. D'Arrigo has written or co-written 37 research publications, sole-authored 3 books, and was sole inventor
on 9 issued patents (in 8 countries). As head of all R&D at Cav-Con Inc., he has served as Principal Investigator on various Nat'l. Inst. of Health
SBIR Phase I, and Phase II, maximum-level grants to Cav-Con Inc. for further development of the Filmix nanoemulsion technology (as applied to
"actively targeted" chemotherapy and/or imaging/monitoring).
SELECTED PUBLICATIONS:
BOOKS:
D'Arrigo, J.S. (2011). Stable Nanoemulsions: Self-Assembly in Nature and Nanomedicine, 436 pp.; Elsevier Science Publishers, Amsterdam and Oxford.
D'Arrigo, J.S. (2003). Stable Gas-in-Liquid Emulsions: Production in Natural Waters and Artificial Media, Second edition, 323 pp.; Elsevier Science Publishers, Amsterdam and New York.
D'Arrigo, J.S. (1986). Stable Gas-in-Liquid Emulsions: Production in Natural Waters and Artificial Media, 220 pp.; Elsevier Science Publishers, Amsterdam and New York.
ARTICLES:
1. Kureshi, I.U., S.Y. Ho, H.C. Onyiuke, A.E. Wakefield, J.S. D'Arrigo, & R.H. Simon. (1999). The affinity of lipid-coated microbubbles to maturing spinal cord injury sites. Neurosurgery 44:1047-1053.
2. Wakefield, A.F., S.Y. Ho, X.G. Li, J.S. D'Arrigo, & R.H. Simon. (1998). The use of lipid-coated microbubbles as a delivery agent for 7ß-hydroxycholesterol to a radiofrequency lesion in the rat brain. Neurosurgery 42:592-598.
3. Ho, S.Y., X.G. Li, A. Wakefield, E. Barbarese, J.S. D'Arrigo, & R.H. Simon. (1997). The affinity of lipid-coated microbubbles for maturing brain injury sites. Brain Res. Bull. 43:543-549.
4. Ho, S.Y., E. Barbarese, J.S. D'Arrigo, C. Smith, & R.H. Simon. (1997). Evaluation of lipid-coated microbubbles as a delivery vehicle for Taxol in tumor therapy. Neurosurgery 40:1260-1268.
5. Barbarese, E., S.Y. Ho, J.S. D'Arrigo, & R.H. Simon. (1995). Internalization of microbubbles by tumor cells in vivo and in vitro. J.Neuro-Oncology 26:25-34.
6. Simon, R.H., S.Y. Ho, D.F. Uphoff, S.C. Lange, & J.S. D'Arrigo. (1993). Applications of lipid-coated microbubble ultrasonic contrast to tumor therapy. Ultrasound in Medicine & Biology 19:123-125.
7. Huang, W., J.C. Grecula, T.M. Button, D.P. Harrington, M.A. Davis, J.S. D'Arrigo, B.H. Laster, & C.S. Springer. (1993). Use of lipid-coated microbubbles (LCM) for susceptibility-based MRI contrast in brain tumors. Proc. 12th Annu. Mttg. Soc. Magnetic Resonance in Med., August 1993, New York, NY.
8. D'Arrigo, J.S., S.Y. Ho, & R.H. Simon. (1993). Detection of experimental rat liver tumors by contrast-assisted ultrasonograpy. Investigative Radiology 28:218-222.
9. D'Arrigo, J.S., & T. Imae. (1992). Physical characteristics of ultrastable lipid-coated microbubbles. J. Colloid & Interface Sci.149:592-595.
10. D'Arrigo, J.S., R.H. Simon, & S.Y. Ho. (1991). Lipid-coated uniform microbubbles for earlier sonographic detection of brain tumors. J. Neuroimaging 1:134-139.
11. D'Arrigo, J.S. (1991). Contrast-assisted tumor detection. Drug News & Perspectives 4:164-167.