Preclinical In-Vivo Safety and Toxicokinetics of D-PLEX100 in an Abdominal Surgery Incision Model in Juvenile Miniature Swine.

D-PLEX100 (D-PLEX) is a novel product candidate made of a polymer-lipid-based matrix (PLEX platform) which contains doxycycline that is being released at a constant rate for 30 days. D-PLEX was developed to prevent surgical site infections, which are a major global health challenge. Previous studies have shown its safety in adult humans, adult swine, and adult rabbits. The aim of this study was to assess the toxicity and safety of D-PLEX also in juvenile animals to support future clinical trials in pediatric patients. Yucatan miniature swine were selected as a model, primarily due to their relatively larger mass. D-PLEX or placebo (formulation without doxycycline) was administered locally to abdominal incisions, and the animal's safety parameters were followed for 9 months and compared to sham-control swine. There was no evidence of any systemic safety concern or local toxicity at the incision site in D-PLEX-treated animals. D-PLEX was detected after 1 month and was fully resorbed at the 3-month time point. The surgical incision sites were fully healed at the 6-month time point in all D-PLEX-treated animals. Toxicokinetic (TK) assessments revealed that doxycycline exhibited low Cmax and therefore minimal systemic exposure following a single dose of local administration. This study provides evidence for the safety of D-PLEX and PLEX-based formulation in juvenile miniature swine and supports its further testing in clinical pediatric population. In addition, it can be used as a reference for future preclinical studies aiming to evaluate the safety of other PLEX-based product candidates for the pediatric population.

Potent antibacterial activity in surgical wounds with local administration of D-PLEX100.

Despite significant advances in infection control guidelines and practices, surgical site infections remain a substantial cause of morbidity, prolonged hospitalization, and mortality. The most effective component of SSI reduction strategies is the preoperative administration of intravenous antibiotics; however, systemic antibiotics drug exposure diminishes rapidly and may result in insufficient prophylactic activity against susceptible and resistant SSI pathogens at the wound. D-PLEX100 (D-PLEX) is an antibiotic-releasing drug (doxycycline) that is supplied as a sterile powder for paste reconstitution with sterile saline. D-PLEX paste is administered locally into the incision site along the entire length of soft tissue and sternal bone wound surfaces prior to skin closure. A single D-PLEX administration is intended for 30 days of constant antimicrobial prophylaxis in the prevention of incisional SSIs. We evaluated D-PLEX minimal bactericidal concentration (MBC) against a panel of bacteria that is prevalent in the abdominal wall and sternal surgical procedures including doxycycline susceptible and resistant strains. D-PLEX in vivo efficacy was assessed in incisional infection rabbit models (abdominal wall and sternal) challenged with a similar bacterial panel. The D-PLEX drug exposure profile was determined by in vitro release assay, and in vivo by quantitative pharmacokinetic parameters of local and systemic doxycycline concentrations released from D-PLEX after local administration in incisional rabbit models. Analyses of pathogens and variations in antibiotic resistance from wound isolates were determined from patients who participated in a previously reported prospective randomized trial that assessed the SSI rate in D-PLEX plus standard of care (SOC) versus SOC alone in colorectal resection surgery. The D-PLEX MBC values demonstrated >3- Log10 reduction in all the organisms tested relative to untreated controls, including doxycycline-resistant bacteria (i.e., Methicillin-resistant Staphylococcus aureus (MRSA), K. pneumoniae, and P. aeruginosa). In vivo, D-PLEX significantly reduced the bacterial loads in all the bacteria tested in both animal models (p=0.0001) with a marked impact observed in E. Coli (>6.5 Log10 reduction). D-PLEX exhibited a zero-order release kinetics profile in vitro for 30 days (R2 = 0.971) and the matched in vivo release profile indicated a constant local release of protein-unbound doxycycline for 30 days at 3-5 mcg/mL with significantly lower (>3 orders of magnitudes) systemic levels. In colorectal surgery patients, where significant SSI reduction was observed, analysis of the positive cultures in the overall population indicated similar pathogen diversity and antibiotic resistance rates in both treatment arms. However, almost all the patients with positive culture in the SOC arm were adjudicated as SSI (94%) compared to only 28% in the D-PLEX arm. The SSI-adjudicated D-PLEX patients also exhibited lower resistance rates to the SOC antibiotics and to MDRs compared to patients in the SOC arm. Thus, D-PLEX provides safe and effective prophylaxis activity against the most prevalent SSI pathogens including doxycycline-susceptible and resistant bacteria. Our findings suggest that D-PLEX is a promising addition to SSI prophylactic bundles and may address the gaps in current SSI prophylaxis. D-PLEX is now evaluated in Phase 3 clinical trial.

D-PLEX100 in an Abdominal Surgery Incision Model in Miniature Swine: Safety Study.

Surgical site infections (SSIs) are a common surgical-related complication. To avoid these complications, a new biodegradable polymer-lipid encapsulation matrix that provides controlled release of doxycycline (doxycycline/polymer-lipid encapsulation matrix [D-PLEX]) has been developed. The aim of this comprehensive study was to evaluate the potential safety of D-PLEX100 in abdominal surgical site. D-PLEX100 was administered into incisions of abdominal surgical site in Yucatan miniature swine, which were followed for up to 6 months and compared to sham-control swine. The D-PLEX100 mass did not migrate from the incisional site, and there was no evidence for systemic toxicity or other safety concerns. Surgical incision sites, including the peritoneal surface, were fully healed at 6 months in all animals. Most of the D-PLEX100 mass was absorbed during the first 3 months, and by 6 months, D-PLEX100 was fully absorbed. Toxicokinetic evaluation revealed that doxycycline concentrations were evident at 30 minutes and persisted to 8 days (71 mg/kg) or at least 15 days (284 mg/kg) and were no longer present in plasma by day 29. This study supports the safety of D-PLEX100 and its favorable degradability profile. A clinical study is being performed to assess the safety and the efficacy of D-PLEX100 to prevent human abdominal SSIs.

Toxicity and Safety Study of D-PLEX100 in a Sternal Surgical Defect in New Zealand White Rabbits.

Bacterial infections are a common complication after surgical procedures. Therefore, local delivery of antibiotics has been developed, including the use of biodegradable polymers. A newly developed product for prevention of surgical site infections is a polymer-lipid encapsulation matrix loaded with doxycycline, named D-PLEX100 (D-PLEX). We evaluated the toxicity and safety of D-PLEX using a sternal surgical defect model in rabbits. D-PLEX was tested with three different concentrations of doxycycline in comparison to sham-operated control after administration into the sternal surgical defect and on the ventral side of the sternum in New Zealand White (NZW) rabbits, following 15 months of exposure. No mortality or abnormal clinical findings were attributed to D-PLEX, and clinical pathology assays were normal. Histological examinations revealed no treatment-related adverse findings in any of the examined tissues, including the osseous and surrounding soft tissues. It has been shown that D-PLEX gradually degraded until complete disappearance after 9 months, and mainly during the first 3 months, in parallel to normal bone formation. In addition, the administration of D-PLEX did not affect sternal bone strength. This study adds to the growing data on preclinical safety studies utilizing biodegradable materials and provides information on the expected normal reaction to biodegradable materials in the sternum of NZW rabbits.

Targeting natural killer cell reactivity by employing antibody to NKp46: implications for type 1 diabetes.

Natural killer (NK) cells belong to the innate lymphoid cells. Their cytotoxic activity is regulated by the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells. We previously reported that the NKp46 receptor is involved in the development of type 1 diabetes (T1D). Subsequently, we hypothesized that blocking this receptor could prevent or hinder disease development. To address this goal, we developed monoclonal antibodies for murine NKp46. One mAb, named NCR1.15, recognizes the mouse homologue protein of NKp46, named Ncr1, and was able to down-regulate the surface expression of NKp46 on primary murine NK cells following antibody injection in vivo. Additionally, NCR1.15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, non-obese diabetic mice and low-dose streptozotocin. Our results show a significantly lower incidence of diabetic mice in the NCR1.15-treated group compared to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests a novel treatment strategy for early insulitis.

Dimerization of NKp46 receptor is essential for NKp46-mediated lysis: characterization of the dimerization site by epitope mapping.

NKp46 is a primary activating receptor of NK cells that is involved in lysis of target cells by NK cells. Previous studies showed that the membrane-proximal domain of NKp46 (NKp46D2) retained the binding of NKp46 to its ligands and is involved in lysis. We studied NKp46D2 by using a peptide-based epitope mapping approach and identified an NKp46D2-derived linear epitope that inhibited NKp46-mediated lysis. The epitope, designated as pep4 (aa 136-155), interacted with NKp46, and lysis by NK cells was inhibited by the presence of pep4. Through modeling and mutagenesis, we showed that pep4 could be involved in NKp46 homodimerization. R145 and D147 contribute to the function of pep4, and R145Q mutation in recombinant NKp46 reduced its binding to target cells. At the cellular level, fluorescent resonance energy transfer analysis revealed that pep4 is indeed involved in dimerization of cell membrane-associated NKp46. We suggest that the NKp46-derived pep4 site is part of the dimerization surface of NKp46 and that NKp46 dimerization contributes to NKp46-mediated lysis by NK cells.

Orally active, antimetastatic, nontoxic diphenyl ether-derived carbamoylphosphonate matrix metalloproteinase inhibitors.

Seven 4-phenoxybenzenesulfonamidopolymethylene carbamoylphosphonates (CPOs) bearing two to eight methylene units in the polymethylene chain were synthesized and evaluated as matrix metalloproteinase (MMP) inhibitors. The five lowest homologues [(CH2)2-6] are selective MMP-2 inhibitors, whereas the two with the longest linkers [(CH2)7,8] lack inhibitory activity. The most potent homologues are those with (CH2)5,6; these two were evaluated for antimetastatic activity in a murine melanoma model and showed good potency both by oral and intraperitoneal administration without any toxic--including musculoskeletal--side effects. In contrast to the previously reported cis-ACCP, which was shown to inhibit MMP-2 for ∼30 min, the new compounds inhibit MMP activity for the duration of measurement, lasting several hours. Pharmacokinetic evaluation revealed, on the one hand, low oral bioavailability; on the other hand, a relatively large calculated volume of distribution, consistent with the observed reversible absorption of CPO 5 to hydroxyapatite, as a model for bone.

Multiple mechanisms downstream of TLR-4 stimulation allow expression of NKG2D ligands to facilitate macrophage/NK cell crosstalk.

The activating receptor NKG2D recognizes proteins that are not normally expressed at the surface of most cells but are expressed during a cellular "stress" response (e.g., upon induction of the DNA damage pathway). This establishes recognition of "induced self" as an important strategy for surveillance of infections or tumor transformation. However, NKG2D ligands can also be induced on human macrophages by TLR stimulation, which has been far less studied. In this paper, we clarify that LPS, which ligates TLR-4, preferentially upregulated MICA and not MICB; CL097, which ligates TLR-7/8, upregulated both MICA and MICB; and polyinosinic-polycytidylic acid, which ligates TLR-3, upregulated neither. To probe how LPS stimulation triggers MICA expression, we determined that the stability of MICA mRNA was much longer than that of MICB mRNA, but neither was changed by LPS stimulation. This finding suggests that increased levels of MICA mRNA following LPS stimulation resulted from increased transcription. However, it was not sufficient for surface protein expression, which was controlled posttranscriptionally via a separate pathway involving the ataxia telangiectasia mutated/ataxia telangiectasia and Rad3 related kinases. Moreover, LPS stimulation decreased expression of microRNAs (miRNA)--miR-17-5, miR-20a, and miR-93--which target MICA, implicating a novel role for miRNAs in NKG2D ligand expression. Thus, TLR stimulation allows expression of NKG2D ligands through multiple pathways, including downmodulation of specific miRNAs.

CD44 is highly expressed on milk neutrophils in bovine mastitis and plays a role in their adhesion to matrix and mammary epithelium.

Mastitis, inflammation of the mammary gland, is a common and economically important disease in dairy animals. Mammary pathogenic organisms, such as Escherichia coli, invade the teat canal,milk ducts, and mammary alveolar space, replicate in mammary secretions, and elicit a local inflammatory response characterized by massive recruitment of blood polymorphonuclear neutrophil leukocytes (PMN) into the alveoli and milk ducts. CD44 is a trans-membrane glycoprotein previously shown to play a role in mediation and control of blood PMN recruitment in response to inflammatory signals. Here we show, for the first time, increased expression of CD44 on recruited milk PMN in bovine mastitis and the expression of a CD44 variant, CD44v10, on these PMN. Furthermore, we demonstrate that CD44 mediates specific adhesion of bovine blood PMN to hyaluronic acid and mammary epithelial cells. Our results suggest that in mastitis CD44 plays a role in recruiting blood PMN into the mammary glands, the exact nature of this role needs to be elucidated.

CD44 involvement in autoimmune inflammations: the lesson to be learned from CD44-targeting by antibody or from knockout mice.

CD44 is a multistructural and multifunctional glycoprotein, the diversity of which is generated by alternative splicing. In this communication we review some aspects related to CD44 structure and function in experimental autoimmune inflammation, focusing on research performed in our own laboratory. We have found that CD44 targeting by antibody, passively injected into DBA/1 mice with collagen-induced arthritis (CIA) and NOD mice with type I diabetes or actively generated by CD44 cDNA vaccination of SJL/j mice with autoimmune encephalomyelitis, markedly reduced the pathological manifestations of these diseases by attenuating cell migration of the inflammatory cells and/or by their apoptotic killing. However, genetic deletion of CD44 by knockout technology enhanced the development of CIA because of molecular redundancy mediated by RHAMM (a receptor of hyaluronan-mediated motility). The mechanisms that stand behind these findings are discussed.

Intercellular transfer of carcinoembryonic antigen from tumor cells to NK cells.

The inhibition of NK cell killing is mainly mediated via the interaction of NK inhibitory receptors with MHC class I proteins. In addition, we have previously demonstrated that NK cells are inhibited in a class I MHC-independent manner via homophilic carcinoembryonic Ag (CEA) cell adhesion molecules (CEACAM1)-CEACAM1 and heterophilic CEACAM1-CEA interactions. However, the cross-talk between immune effector cells and their target cells is not limited to cell interactions per se, but also involves a specific exchange of proteins. The reasons for these molecular exchanges and the functional outcome of this phenomenon are still mostly unknown. In this study, we show that NK cells rapidly and specifically acquire CEA molecules from target cells. We evaluated the role of cytotoxicity in the acquisition of CEA and demonstrated it to be mostly killing independent. We further demonstrate that CEA transfer requires a specific interaction with an unknown putative NK cell receptor and that carbohydrates are probably involved in CEA recognition and acquisition by NK cells. Functionally, the killing of bulk NK cultures was inhibited by CEA-expressing cells, suggesting that this putative receptor is an inhibitory receptor.

CD44 variant DNA vaccination with virtual lymph node ameliorates experimental autoimmune encephalomyelitis through the induction of apoptosis.

Standard CD44 (CD44s) and its alternatively spliced variants (CD44v) were found to be associated with the metastatic potential of tumor cells, and with cell migration of autoimmune inflammatory cells, including cells involved in experimental autoimmune encephalomyelitis (EAE). The aim of the present study was to evaluate whether induction of anti-CD44 immune reactivity, through cDNA vaccination could down-regulate EAE. Our vaccination technique involved the insertion of CD44s or CD44v cDNA into a silicone tube filled with 2.5 cm long segment of hydroxylated-polyvinyl acetate wound dressing sponge (forming a virtual lymph node) which was implanted under the skin of SJL/J mice immunized with myelin antigens for EAE induction. Animals vaccinated with CD44v cDNA developed significantly less severe EAE when compared with sham vaccinated animals or animals vaccinated with CD44s cDNA. The in vitro proliferation of lymphocytes was preserved regarding myelin antigens and mitogens. Histopathological examinations revealed a significant reduction of EAE lesions and enhanced apoptosis in central nervous system (CNS)-infiltrating cells of the successfully vaccinated animals. Such methods of cDNA vaccination with CD44 could be applicable in inflammatory CNS diseases, like multiple sclerosis.

Expression of extra trinucleotide in CD44 variant of rheumatoid arthritis patients allows generation of disease-specific monoclonal antibody.

Selective targeting of cells engaged in pathological activities is a major challenge for medical research. We generated monoclonal antibodies (mAbs) that exclusively bind, at concentrations ranging from 2 to 100 microg/ml, to a modified CD44 variant (designated CD44vRA) expressed on synovial fluid cells from joints of rheumatoid arthritis (RA) patients. These mAbs cross-reacted with keratinocytes expressing wild type CD44vRA (CD44v3-v10) only at a relatively high concentration (200 microg/ml). Sequence analysis of CD44vRA cDNA revealed, in 33 out of 43 RA and psoriatic arthritis patients, an extra intron-derived trinucleotide, CAG, which allows translation of an extra alanine. This insertion imposes a configurational change on the cell surface CD44 of RA synovial fluid cells, creating an immunogenic epitope and potentiating the ability to produce disease-specific antibodies. Indeed, the anti-CD44vRA mAbs (designated F8:33) were able to induce apoptosis in synovial fluid cells from RA patients, but not in peripheral blood leukocytes from the same patients, in keratinocytes from normal donors or in synovial fluid cells from osteoarthritis patients. Furthermore, injection of anti-CD44vRA mAbs reduced joint inflammation in DBA/1 mice with collagen-induced arthritis. These findings show that anti-CD44vRA mAbs are both bioactive and RA-specific.

Reciprocal regulation of human natural killer cells and macrophages associated with distinct immune synapses.

Natural killer (NK) cells directly lyse tumor or viral-infected cells but also an important role for NK cell cytotoxicity in regulating the extent of immune responses is emerging. Here, we show that autologous human macrophages activated NK cell proliferation and cytokine secretion, increased expression of activating receptors, and primed NK cell cytotoxicity against susceptible target cells. Ligation of NK cell 2B4, and not NKp30 (known to be important for DC-mediated NK cell activation), is critical for this macrophage-mediated NK cell activation. Reciprocally, however, NK cells regulated macrophage activity by directly killing macrophages stimulated by high doses of LPS. Cytolysis was triggered by NKG2D recognition of stress-inducible class I major histocompatibility complex (MHC)-like ligands on macrophages: high doses of LPS induced transcription and surface expression of ULBP1, ULBP2, and ULBP3 and surface expression of constitutively transcribed MICA. Thus, these data suggest a new function for NK cell cytotoxicity in eliminating overstimulated macrophages. Additionally, these interactions define, for the first time, 2 distinct activating NK cell synapses: lytic and nonlytic. Triggering NK cell proliferation and cytokine secretion, but not cytolysis, specifically associated with synaptic accumulation of macrophage F-actin and NK cell 2B4, while macrophages were killed when NK cell F-actin and macrophage ICAM-1 accumulated around a central cluster of NK cell NKG2D/DAP10.

Increased surveillance of cells in mitosis by human NK cells suggests a novel strategy for limiting tumor growth and viral replication.

The threat from cancer cells is inherently linked to cell-cycle progression, and viral genomes commonly replicate, for example, within episomes or proviruses, during mitosis. We report here that human natural killer (NK) cells bound cells in mitosis and attacked pathogenic cells in mitosis more effectively than the same cells in other stages of the cell cycle. Thus, cells in mitosis warrant and undergo heightened surveillance, a novel strategy for immunologic assessment of danger. Recognition of cells in mitosis involved ligation of activating NK-cell receptors and binding to target-cell hyaluronan, a component of the pericellular matrix known to be increased during mitosis. Direct interaction between activating NK-cell receptors and hyaluronan is possible, but other mechanisms consistent with our data are also discussed.

Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria.

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > approximately 0.7 microm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at approximately 1 microm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

Long-distance calls between cells connected by tunneling nanotubules.

Long membrane tethers between cells, known as membrane nantotubes or tunneling nanotubules, create supracellular structures that allow multiple cell bodies to act in a synchronized manner. Calcium fluxes, vesicles, and cell-surface components can all traffic between cells connected by nanotubes. Thus, complex and specific messages can be transmitted between multiple cells, and the strength of signal will suffer relatively little with the distance traveled, as compared to the use of soluble factors to transmit messages. Connecting multiple antigen-presenting cells, for example, can help amplify and coordinate immune responses that are distal to an antigenic site. Conversely, because the ability of a pathogen to spread between cells is a key determinant of its capacity to multiply, pathogens may exploit nanotubes for their own transmission.

The mechanism of molecular redundancy in autoimmune inflammation in the context of CD44 deficiency.

Molecular redundancy refers to the ability of genes to back up damaged genes or gene loss. Although this term is widely discussed in many scientific circles, the process is still ill-defined, as shown by reviewing examples from the literature. Exploring the collagen-induced arthritis model in the context of CD44 knockout mice, we suggest a mechanistic explanation for molecular redundancy that depends neither on upregulation of the compensating molecule nor on structural similarity between the original molecule and the replacement molecule. The backup process is dependent, however, on two key properties shared by the two molecules: ligand binding and support of cell trafficking.

RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: a different interpretation of redundancy.

We report here that joint inflammation in collagen-induced arthritis is more aggravated in CD44-knockout mice than in WT mice, and we provide evidence for molecular redundancy as a causal factor. Furthermore, we show that under the inflammatory cascade, RHAMM (receptor for hyaluronan-mediated motility), a hyaluronan receptor distinct from CD44, compensates for the loss of CD44 in binding hyaluronic acid, supporting cell migration, up-regulating genes involved with inflammation (as assessed by microarrays containing 13,000 cDNA clones), and exacerbating collagen-induced arthritis. Interestingly, we further found that the compensation for loss of the CD44 gene does not occur because of enhanced expression of the redundant gene (RHAMM), but rather because the loss of CD44 allows increased accumulation of the hyaluronic acid substrate, with which both CD44 and RHAMM engage, thus enabling augmented signaling through RHAMM. This model enlightens several aspects of molecular redundancy, which is widely discussed in many scientific circles, but the processes are still ill defined.

Cutting edge: Membrane nanotubes connect immune cells.

We present evidence that nanotubular highways, or membrane nanotubes, facilitate a novel mechanism for intercellular communication in the immune system. Nanotubes were seen to connect multiple cells together and were readily formed between a variety of cell types, including human peripheral blood NK cells, macrophages, and EBV-transformed B cells. Nanotubes could be created upon disassembly of the immunological synapse, as cells move apart. Thus, nanotubular networks could be assembled from transient immunological synapses. Nanotubes were seen to contain GFP-tagged cell surface class I MHC protein expressed in one of the connected cells. Moreover, GPI-conjugated to GFP originating from one cell was transferred onto the surface of another at the connection with a nanotube. Thus, nanotubes can traffic cell surface proteins between immune cells over many tens of microns. Determining whether there are physiological functions for nanotubes is an intriguing new goal for cellular immunology.