SQ Route Research Articles

Isoproterenol induced cardiac hypertrophy: A comparison of three doses and two delivery methods in C57BL/6J mice.

Heart Failure (HF) continues to be a complex public health issue with increasing world population prevalence. Although overall mortality has decreased for HF and hypertrophic cardiomyopathy (HCM), a precursor for HF, their prevalence continues to increase annually. Because the etiology of HF and HCM is heterogeneous, it has been difficult to identify novel therapies to combat these diseases. Isoproterenol (ISP), a non-selective β-adrenoreceptor agonist, is commonly used to induce cardiotoxicity and cause acute and chronic HCM and HF in mice. However, the variability in dose and duration of ISP treatment used in studies has made it difficult to determine the optimal combination of ISP dose and delivery method to develop a reliable ISP-induced mouse model for disease. Here we examined cardiac effects induced by ISP via subcutaneous (SQ) and SQ-minipump (SMP) infusions across 3 doses (2, 4, and 10mg/kg/day) over 2 weeks to determine whether SQ and SMP ISP delivery induced comparable disease severity in C57BL/6J mice. To assess disease, we measured body and heart weight, surface electrocardiogram (ECG), and echocardiography recordings. We found all 3 ISP doses comparably increase heart weight, but these increases are more pronounced when ISP was administered via SMP. We also found that the combination of ISP treatment and delivery method induces contrasting heart rate, RR interval, and R and S amplitudes that may place SMP treated mice at higher risk for sustained disease burden. Mice treated via SMP also had increased heart wall thickness and LV Mass, but mice treated via SQ showed greater increase in gene markers for hypertrophy and fibrosis. Overall, these data suggest that at 2 weeks, mice treated with 2, 4, or 10mg/kg/day ISP via SQ and SMP routes cause similar pathological heart phenotypes but highlight the importance of drug delivery method to induce differing disease pathways.

Subcutaneous checkpoint inhibition is equivalent to systemic delivery when combined with nelitolimod delivered via pressure-enabled drug delivery for depletion of intrahepatic myeloid-derived suppressor cells and control of liver metastases

BackgroundToll-like receptor 9 (TLR9) agonists induce inflammatory responses that promote the killing of infectious micro-organisms, cancer cells and develop adaptive immune responses. Their ability as immunomodulators to enhance the activity...

Switching Biologics from IV to SQ Routes

Switching Biologics from IV to SQ Routes

Subcutaneous Administration of Medications and Fluids by Nonprofessional Caregivers at Home.

Background: Patients requiring home-based palliative care have advanced complex illnesses with functional limitations and decline. This retrospective study reviewed caregiver administration of subcutaneous (SQ) medications and fluids when symptom control could not be achieved using the oral route. Methods: Medical records from September 1, 2017 to February 28, 2018 were reviewed for 272 consecutive patients who received SQ administration of medications or fluids at a home-based palliative care program. We analyzed the clinical characteristics of patients and caregivers, medications administered, and catheter outcomes. Results: Patients' median age was 74 years, and 163 (60%) were women. The most common cancer diagnoses were stomach 26 (12%), lung 22 (10%), and colorectal 20 (9%). Dementia 24 (44%), cerebrovascular disease 9 (16%), and congestive heart failure 7 (13%) were the most frequent nonmalignant diseases. Poor symptom control 162 (60%) and impaired oral intake 107 (39%) were the most common indications for an SQ route of administration. Nonprofessional caregivers trained by a nurse administered medications to 218 patients (80%). During interventions, the patients received a mean of 4 medications (±2 standard deviation). A total of 903 catheters were inserted, 15/732 (2%) catheters handled by nonprofessional caregivers caused a local infection, compared with 3/171 (1.8%) of catheters handled by nurses. Hydromorphone was the most common opioid used (57%), followed by morphine (35%). The median length of stay in the program was 24 days (interquartile range: 11-60). Conclusions: SQ administration of medications and fluids by nonprofessional caregivers trained by health care professionals is feasible and promising, but additional testing is needed.

Gonadotropin-releasing hormone analog therapies for children with central precocious puberty in the United States.

Gonadotropin-releasing hormone agonists (GnRHa's) are the standard treatment for children with central precocious puberty (CPP). We aim to present data on available GnRHa options with an easy-to-review table and discuss factors that influence treatment selection. Five GnRHa's are currently FDA-approved and prescribed in the US and published data suggest similar safety and efficacy profiles over the first year of treatment. One- and 3-month intramuscular (IM) leuprolide acetate (LA) have long-term safety and efficacy data and allow for flexible dosing. Six-month IM triptorelin pamoate offers a longer duration of treatment, but without long-term efficacy and outcome data. Six-month subcutaneous (SQ) LA combines a SQ route of injection and long duration of action but lacks long-term efficacy and outcome data. The 12-month SQ histrelin acetate implant avoids injections and offers the longest duration of action, but requires a minor surgical procedure with local or general anesthesia. Factors in treatment selection include route of administration, needle size, injection volume, duration of action, and cost. The current GnRHa landscape provides options with varying benefits and risks, allowing physicians and caregivers to select the most appropriate therapy based on the specific needs and concerns of the child and the caregiver. Agents have different advantages and disadvantages for use, with no one agent displaying superiority.

Pharmacokinetics of Single-Dose Intramuscular and Subcutaneous Injections of Buprenorphine in Common Marmosets (Callithrix jacchus).

Although buprenorphine is the most frequently used opioid analgesic in common marmosets (Callithrix jacchus), there is limited information in the literature supporting current dosing regimens used for this species. The purpose of this study was to determine the pharmacokinetic profiles of single-dose buprenorphine HCl administered intramuscularly (IM) at 0.01 mg/kg in 6 adult marmosets (1.8 to 12.8 y old; 2 males, 4 females) and subcutaneously (SQ) at 0.01 mg/kg in 6 adult marmo- sets (2.3-4.4 y old; 3 males, 3 females) by mass spectrometry. Blood was collected at multiple time points from 0.25 to 24 h from unsedated animals following a hybrid sparse-serial sampling design. The maximal observed plasma concentration of buprenorphine (Cmax ) administered IM (2.57 ± 0.95 ng/mL) was significantly higher than administered SQ (1.47 ± 0.61 ng/mL). However, the time to Cmax (Tmax) was not statistically different between routes (17.4 ± 6 min for IM and 19.8 ± 7.8 min for SQ). The time of the last quantifiable concentration of buprenorphine was 5 ± 1.67 h for IM compared with 6.33 ± 1.51 h for SQ, which was not statistically different. The mean buprenorphine plasma concentration-time curves were used to propose a dosing frequency of 4 to 6 h for buprenorphine at 0.01 mg/kg IM or SQ based on a theoretical therapeutic plasma concentration threshold of 0.1 ng/mL. Based on the mean pharmacokinetic parameters and plasma-concentration time curves, both IM and SQ routes of buprenorphine at this dose provide a rapid increase in the plasma concentration of buprenorphine above the therapeutic threshold, and may be more effective for acute rather than long-lasting analgesia. Further studies are needed to examine repeated dosing regimens and the efficacy of buprenorphine in common marmosets.

First-in-Human Clinical Trial of the IL-15 Super-Agonist Complex ALT-803 for Patients Relapsed after Allogeneic Hematopoietic Cell Transplantation

Background : The prognosis of hematologic malignancy patients who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) remains poor and an unmet clinical need. IL-15 is a cytokine that stimulates both CD8 T cells and NK cells without significant effects on Tregs. ALT-803 is an IL-15/IL15Rα-Fc super-agonist complex with enhanced stability in vivo. We hypothesized that ALT-803 may augment T and NK cell number and function in the post allo-HCT relapse setting without Treg modulation, and initiated first-in-human phase I dose escalation clinical trial to test this idea. We previously reported preliminary results of the IV cohort (Miller JS et al, ASH 2016); here we report the complete study.Methods : Patients with hematologic malignancies that have relapsed >60 days after allo-HCT with no active graft versus host disease (GVHD), off immune-suppression, and with at least 10% donor chimerism were eligible. A phase 1 dose finding study design used a 3x3 dose escalation to define the maximal tolerated dose, followed by an expansion cohort. ALT-803 was administered to N=33 patients in 4 weekly doses via two routes: IV or SQ (Table 1). Median age was 58 (22-74) years, diagnoses primarily included AML (67%) and MDS (18%), and type of donor included matched-related (39%), matched-unrelated (21%), cord blood (27%), or related haploidentical (12%). 27% of patients had any grade of GVHD with <1% having a prior history of aGVHD > grade II, or moderate to severe cGVHD. In the IV cohort (N=16) 4 dose levels of ALT-803 were administered: 1 (n=6), 3 (n=3), 6 (n=4), and 10 (n=3) mcg/kg. In a second SQ cohort, 17 patients were treated at two dose levels, 6 (n=8) and 10 (n=9) mcg/kg. 4 patients in the SQ cohort were not evaluable and were replaced. Immunomonitoring was performed using flow and mass cytometry to evaluate changes in immune cells, with a focus on T and NK cells.Results: ALT-803 was well tolerated in both the IV and SQ cohorts; no DLTs were observed. The most common AEs were constitutional symptoms. Injection site rash occurred commonly in the SQ patients, which were self-limiting and showed a variable degree of CD8+, CD4+ T cells and NK cells on biopsies. No patients developed cytokine release or capillary leak syndromes, nor was there new onset severe GVHD or clinical progression of prior GVHD. 3 patients had grade 4 or 5 severe adverse events: sepsis (n=2) and an intracranial hemorrhage, which were expected in this high-risk patient HCT population with active disease. 3 clinical responses occurred with one CR and two patients with stable disease. For the CR (6 mcg/kg IV cohort), the patient's BM blast percentage decreased from 9% to 3%, his previously complex cytogenetics with -7 normalized, and his donor chimerism increased from 46% to 93%. Pharmacokinetic analyses revealed a longer half-life in SQ (10 mcg/kg: T1/2 29.3+10.3 h) vs. IV (10 mcg/kg: T-1/2 mean 4.87+3.46h) (Figure 1A). There was marked induction of Ki67+ NK cells peaking at day 5 post-injection, with significantly longer persistence of Ki67+ via the SQ route (10 mcg/kg, IV: 32%, SQ 70%, P=0.002). Increased NK cell percentages (Figure 1B) and absolute numbers were observed (Figure 1C). Ki67 was also induced within CD8+ T cells, but to a lesser degree than in the NK cells. Minimal induction of Ki67 was observed within CD4+ T cells. Mass cytometry was performed to deeply immunophenotype the NK cell compartment (Romee R et al, Sci Transl Med, 2016) of N=4 patients (10 mcg/kg SQ), comparing pre-, days 5 and 7 after the first dose of ALT-803. viSNE clustering of 37 different NK cell markers revealed marked alteration of the NK cell compartment after ALT-803. There were minimal changes in KIR and NKG2A expression on NK cells, but consistent upregulation of the receptors NKG2D, NKp30, and TIM-3 in vivo. There was more inter-patient variability with other receptors, with LAG-3 and DNAM-1 each being markedly upregulated in 1 of 4 patients evaluated.Conclusions: ALT-803 was a safe, well-tolerated immunostimulatory agent when administered in the post-HCT setting, which resulted in 1 CR and SD in several patients with AML and MDS. We identified distinct PK profiles following IV vs. SQ administration, and evidence of ALT-803 stimulation of the NK cell and CD8 T cell compartments, without major changes in CD4+ T cells. ALT-803 is a promising agent that warrants additional investigation in the allo-HCT setting, and in combination with other immunotherapy strategies. [Display omitted] DisclosuresJeng:Altor Bioscience: Employment, Equity Ownership. Rock:Altor Bioscience Corp: Employment. Wong:Altor BioScience: Employment, Equity Ownership. Miller:Oxis Biotech: Consultancy; Celegene: Consultancy; Fate Therapeutics: Consultancy, Research Funding.

Inhibit Clinical Trials Platform to Prevent and Eradicate Inhibitors: Feasibility Survey of Current Prophylaxis and Immune Tolerance Practices

Inhibit Clinical Trials Platform to Prevent and Eradicate Inhibitors: Feasibility Survey of Current Prophylaxis and Immune Tolerance Practices

Eliminating Fc N-Linked Glycosylation and Its Impact on Dosing Consideration for a Transferrin Receptor Antibody-Erythropoietin Fusion Protein in Mice.

Erythropoietin (EPO), a hematopoietic growth factor and a promising therapy for Alzheimer's disease, has low permeability across the blood-brain barrier. The transferrin receptor antibody fused to EPO (TfRMAb-EPO) is a chimeric monoclonal antibody that ferries EPO into the brain via the transvascular route. However, TfRMAbs have Fc-effector function-related adverse effects including reticulocyte suppression. To overcome this, we recently developed an effectorless TfRMAb-EPO fusion protein, designated TfRMAb-N292G-EPO, by eliminating the Fc N-linked glycosylation site at position 292 of the antibody heavy chain. The mutant fusion protein showed enhanced plasma clearance and dramatically reduced plasma concentrations compared with the wild-type (WT) nonmutant fusion protein. This increased clearance of the aglycosylated TfRMAb is expected to increase the injection dose of the mutant fusion protein. To provide a basis for future therapeutic uses of this IgG-neurotrophin fusion protein, the current study aimed to characterize the pharmacokinetic profile of this effectorless TfRMAb-N292G-EPO at different doses following different routes of administration in the mouse. Adult C57BL/6J male mice were injected with a single dose (3, 6, 9, or 20 mg/kg; n = 3-6 per dose) of TfRMAb-N292G-EPO through either the subcutaneous (SQ) or intraperitoneal (IP) route. TfRMAb-N292G-EPO plasma concentrations were determined using an enzyme-linked immunosorbent assay. Mice were sacrificed 24 h after injection, and terminal blood was used for a complete blood count. Brain concentrations in the WT- and mutant fusion protein-treated mice were compared. We observed stark differences in the plasma pharmacokinetics of TfRMAb-N292G-EPO between the IP and SQ routes of administration. Dose escalation from 3 to 20 mg/kg increased the plasma Cmax only 3.5-fold for the SQ route, compared with a 35-fold increase for the IP route. The plasma Cmax was 15.0 ± 2.0, 21.3 ± 4.1, 21.3 ± 6.4, and 52.8 ± 27.9 ng/mL following SQ injection and 288 ± 47, 389 ± 154, 633 ± 194, and 10,066 ± 7059 ng/mL following IP injection for 3, 6, 9, and 20 mg/kg doses, respectively. The plasma Cmax following the SQ route was therefore 19- to 190-fold lower than that following the IP route. This finding is consistent with a 31-fold higher apparent clearance following the SQ route compared with the IP route at the highest dose administered. The brain concentrations in the mice treated with a 3 mg/kg dose of the mutant fusion protein were lower than those in the nonmutant WT-treated mice. No reticulocyte suppression was observed at the 3 mg/kg SQ dose of TfRMAb-N292G-EPO. However, reticulocyte suppression increased with an increase in dose and area under the plasma concentration-time curve (AUC) for both the IP and SQ routes. Overall, elimination of Fc N-linked glycosylation, to mitigate TfRMAb effector function side effects, has a profound effect on the plasma exposure of TfRMAb-N292G-EPO at therapeutic as well as high doses (3-20 mg/kg). This effect is more pronounced following SQ injection. The low plasma concentrations of the mutant fusion protein following a 3 mg/kg dose resulted in negligible brain uptake. The beneficial rescue of reticulocyte reduction by the N292G mutation is a function of AUC and is negated at high doses of the N292G mutant.

Q&amp;A on the Paper of Kurukulasuriya et al. (2017) on IBD Vaccine Efficacy Against a Canadian Variant IBDV Strain in Broiler Chickens

Kurukulasuriya, et al. (2017) are reporting the efficacy of two IBD vaccines against an early (6 days post-hatch) challenge with a variant Canadian IBDV strain in broilers. A modified live vaccine(UNIVAX BD) administered by SQ route at 1 dayof-age delayed infection whereas an HVT-IBD vector vaccine (VAXXITEK HVT+IBD) administered in ovodid not protect. Furthermore, the authors suggested that the HVT-IBD vector induced immunosuppression responsible for an earlier IBDV challenge strain replication in the bursa. The data presented in the paper showed no evidence of VAXXITEK HVT+IBD vaccine take since the mean IBD ELISA antibody titer at D35 in the vaccinated/non-challenged group was not significantly different from that of the non-vaccinated group. It wasmuch lower than the expected one based on previous studies performed in the same conditions : in ovo vaccination of broilers [1,2]. Since there is no evidence of vaccine take, the other potential effects (immunosuppression and earlier IBDV replication in the bursa) observed in that group cannot be attributed to the vaccine. Since its launch in 2006 in Brazil, VAXXITEK HVT+IBD has been licensed in more than 75 countries and more than 80 billion birds have been vaccinated. VAXXITEK HVT+IBD is protecting against a wide variety of IBDV strains including the classical, the very virulent and different variant strains. To our knowledge, noabsence of efficacy nor bursa depletions have been so far officially reported as long as the vaccine has been administered properlyto healthy embryonated eggs or to healthy one-day-old chicks.

First-in-human phase 1 clinical study of the IL-15 superagonist complex ALT-803 to treat relapse after transplantation

AbstractNew therapies for patients with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) are needed. Interleukin 15 (IL-15) is a cytokine that stimulates CD8+ T-cell and natural killer (NK) cell antitumor responses, and we hypothesized this cytokine may augment antileukemia/antilymphoma immunity in vivo. To test this, we performed a first-in-human multicenter phase 1 trial of the IL-15 superagonist complex ALT-803 in patients who relapsed >60 days after allo-HCT. ALT-803 was administered to 33 patients via the IV or subcutaneous (SQ) routes once weekly for 4 doses (dose levels of 1, 3, 6, and 10 μg/kg). ALT-803 was well tolerated, and no dose-limiting toxicities or treatment-emergent graft-versus-host disease requiring systemic therapy was observed in this clinical setting. Adverse events following IV administration included constitutional symptoms temporally related to increased serum IL-6 and interferon-γ. To mitigate these effects, the SQ route was tested. SQ delivery resulted in self-limited injection site rashes infiltrated with lymphocytes without acute constitutional symptoms. Pharmacokinetic analysis revealed prolonged (>96 hour) serum concentrations following SQ, but not IV, injection. ALT-803 stimulated the activation, proliferation, and expansion of NK cells and CD8+ T cells without increasing regulatory T cells. Responses were observed in 19% of evaluable patients, including 1 complete remission lasting 7 months. Thus, ALT-803 is a safe, well-tolerated agent that significantly increased NK and CD8+ T cell numbers and function. This immunostimulatory IL-15 superagonist warrants further investigation to augment antitumor immunity alone and combined with other immunotherapies. This trial was registered at www.clinicaltrials.gov as #NCT01885897.

Immunogenicity and protective efficacy of the norovirus P particle-M2e chimeric vaccine in chickens

The ectodomain of the influenza matrix protein 2 (M2e) is highly conserved across strains and has been shown to be a promising candidate for universal influenza vaccine in the mouse model. In this study, we tested immune response and protective efficacy of a chimeric norovirus P particle containing the avian M2e protein against challenges with three avian influenza (AI) viruses (H5N2, H6N2, H7N2) in chickens. Two-week-old specific pathogen free chickens were vaccinated 3 times with an M2e-P particle (M2e-PP) vaccine via the subcutaneous (SQ) route with oil adjuvant, and transmucosal routes (intranasal, IN; eye drop, ED; microspray, MS) without adjuvant. M2e-PP vaccination via the SQ route induced significant IgG antibody responses which were increased by each booster vaccination. In groups vaccinated via IN, ED or MS, neither IgG nor IgA responses were detected from sera or nasal washes of immunized birds. The M2e-PP vaccination via the SQ route significantly reduced the virus shedding in the trachea and the cloaca for all three challenge viruses. Despite the absence of detectable IgG and IgA responses in birds vaccinated with the M2e-PP via intranasal routes, a similar level of reduction in virus shedding was observed in the IN group compared to the SQ group. Our results supports that the universal vaccine approach using M2e-based vaccine can provide cross-protection against challenge viruses among different HA subtypes although the efficacy of the vaccine should be enhanced further to be practical. Better understanding of the protective immune mechanism will be critical for the development of an M2e-based vaccine in chickens.

Hepatitis B vaccination is effective by subcutaneous route in children with bleeding disorders: a universal data collection database analysis.

Subcutaneous (SQ) vs. intramuscular (IM) vaccination may cause fewer injection site complications in children with bleeding disorders, but little is known about comparative immunogenicity. To compare immunogenicity of hepatitis B virus (HBV) vaccination administered SQ or IM to individuals <2 years old with bleeding disorders, we performed a retrospective analysis of HBV surface antibody titres among patients enrolled in the universal data collection database who had received three doses of HBV vaccine solely by one route (SQ or IM). Data reviewed were from an initial visit before 24 months of age, until time of hepatitis antibody titre testing. The SQ and IM study groups did not differ in demographics, haemophilia type or severity or bleeding history. The mean age at the time of HBV surface antibody (anti-HBs) testing was 56.9 ± 20.3 months. Eighty-five of 92 subjects (92.4%) who received vaccine SQ developed a positive antibody titre (>12 IU/L), compared to 101/114 (88.6%) who received IM (P = 0.30). There was no statistically significant difference in distribution of titre values. The average age of the subjects at time of testing was 53 ± 20 months in the SQ group vs. 60 ± 20 months in the IM group (P = 0.02). The average time between the last dose of vaccine and anti-HBs testing was 47.6 ± 18.5 months among SQ vaccinated subjects vs. 51.6 ± 20.5 months in the IM group (P = 0.2). Immunogenicity to hepatitis B vaccination by the SQ and IM routes is similar.

P.17.8 The co-formulation of pharmacological chaperone AT2220 with recombinant human acid alpha-glucosidase improves enzyme uptake and glycogen reduction in a mouse model of Pompe disease

Pompe disease is an inherited lysosomal storage disease that results from deficiency in acid alpha-glucosidase (GAA) activity, and is characterized by progressive accumulation of lysosomal glycogen in heart and skeletal muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) is the only approved treatment available for Pompe disease. While rhGAA provides some clinical benefits, the infused enzyme tends to be unstable at neutral pH/body temperature, shows insufficient uptake in key tissues, and can elicit immune responses that affect tolerability and efficacy. We have shown previously that oral pre-administration of the pharmacological chaperone AT2220 (1-deoxynojirimycin HCl, duvoglustat HCl) improves the pharmacological properties of rhGAA via binding and stabilization, leading to increased enzyme uptake and glycogen reduction in GAA knock-out (KO) mice. In this study we tested the effects of intravenous (IV) and subcutaneous (SQ) administration of co-formulated AT2220 and rhGAA (AT2220 + rhGAA) as an alternative to giving AT2220 orally prior to rhGAA IV. In rats, IV or SQ administration of co-formulated AT2220 + rhGAA increased the circulating half-life of rhGAA up to twofold, though the maximal rhGAA plasma levels achieved via the SQ route were significantly lower than those seen following IV administration. In GAA KO mice, four IV administrations of co-formulated AT2220 + rhGAA resulted in up to 2.5-fold greater enzyme uptake and glycogen reduction in disease-relevant tissues compared to rhGAA alone; histological staining confirmed reduced skeletal muscle glycogen. Interestingly, four SQ administrations of co-formulated AT2220 + rhGAA increased rhGAA uptake in GAA KO mice, which was not significantly different from those seen following four IV administrations of rhGAA alone. Collectively, these data highlight the potential effects of co-formulated AT2220 + rhGAA using IV or SQ administration, thus warranting further preclinical investigation.

Photocatalytic degradation of 2,4-dichlorophenoxyacetic acid under visible light: Effect of synthesis route

The photocatalytic degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under visible light exposure was studied using TiO2 and TiO2 doped with nitrogen (1% and 5%) that was prepared by the sol–gel (SG) and sonochemistry (SQ) methods. In fact, the sonochemical route represents a sustainable alternative for saving energy during the synthesis. The results show that the half-life time (t1/2) was 71 min for the 5% N–TiO2 photocatalyst prepared by the SQ. According to XRD results, semi-crystalline TiO2 was obtained using both synthetic routes at 400 °C. However, the presence of a dopant using the SG method causes an increase in the crystallinity of the TiO2 materials prepared, and the crystallinity of the SQ route is inhibited at higher nitrogen content. The XPS data showed the presence of nitrogen (398–401 eV) in synthesized TiO2. The results of the band gap energy (Eg) using a Kubelka–Munk transformation indicate that both methods of synthesis show higher absorbances when nitrogen was incorporated into the matrix of TiO2, leading to the decrease of Eg from 3.2 eV to 3.0 eV. The TEM analysis shows that the nanoparticles were obtained with sizing less than 40 nm, and the incorporation of nitrogen causes a decrease in the particle size. Finally, the surface properties are consistent with the observed crystallinity in the materials.

Pharmacokinetics and Brain Uptake of an IgG-TNF Decoy Receptor Fusion Protein Following Intravenous, Intraperitoneal, and Subcutaneous Administration in Mice

Tumor necrosis factor (TNF)-α is a proinflammatory cytokine active in the brain. Etanercept, the TNF decoy receptor (TNFR), does not cross the blood-brain barrier (BBB). The TNFR was re-engineered for BBB penetration as a fusion protein with a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse and mediates transport via the endogenous BBB TfR. To support future chronic treatment of mouse models of neural disease with daily administration of the cTfRMAb-TNFR fusion protein, a series of pharmacokinetics and brain uptake studies in the mouse was performed. The cTfRMAb-TNFR fusion protein was radiolabeled and injected into mice via the intravenous, intraperitoneal (IP), or subcutaneous (SQ) routes of administration at doses ranging from 0.35 to 10 mg/kg. The distribution of the fusion protein into plasma following the IP or SQ routes was enhanced by increasing the injection dose from 3 to 10 mg/kg. The fusion protein demonstrated long circulation times with high metabolic stability following the IP or SQ routes of injection. The IP or SQ routes produced concentrations of the cTfRMAb-TNFR fusion protein in the brain that exceed by 20- to 50-fold the concentration of TNFα in pathologic conditions of the brain. The SQ injection is the preferred route of administration, as the level of cTfRMAb fusion protein produced in the brain is comparable to that generated with intravenous injection, and at a much lower plasma area under the concentration curve of the fusion protein as compared to IP administration.

Phase II Trial of Intravenously Administered AMD3100 (Plerixafor) for Stem Cell Mobilization in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation Following a Lenalidomide-Based Initial Therapy

Abstract 2992 Background:Patients with myeloma receiving initial therapy with lenalidomide-based regimens can have difficulty collecting adequate stem cells for an autologous transplant. Stem cell collection in these patients can be significantly enhanced by addition of the CXCR-4 antagonist plerixafor to the mobilization regimen. Plerixafor is typically given subcutaneously (SQ), with collection approximately 11 hours after injection to obtain maximum yield. Intravenous administration can potentially allow more rapid and predictable mobilization compared to the SQ route. We designed this trial to prospectively assess the efficacy of intravenous plerixafor administration in patients undergoing lenalidomide therapy. Patients and methods:Patients who were receiving initial therapy with a lenalidomide-based regimen and were undergoing stem cell collection within 12 months of their myeloma diagnosis were enrolled. Patients received GCSF at 10 μg/kg/day for 4 days followed by addition of plerixafor at 0.24 mg/kg/dose starting on day 5. Plerixafor was administered intravenously early morning (6–7 am) followed by apheresis beginning 4–5 hours later. Plerixafor was administered for a maximum of 4 days; but patients could continue apheresis beyond the 4th day at treating physician discretion. The aims of the study were to determine the proportion of patients reaching a stem cell yield of at least 3 million CD34 cells/kg by second day of apheresis, the safety and tolerability of intravenously administered plerixafor, and the overall rate of failure to mobilize (defined as less than 2.5 million CD34 cells/kg in 4 collections). Results:Thirty-seven patients were accrued between December 2009 – April 2011, and 36 were eligible for analysis. The median age was 61 years (range; 28–73); 61% were male. The median time from start of initial therapy to enrollment was 4.6 months (range; 2.6 to 11.1) and the median cycles of lenalidomide were 4 (range; 3–11). Thirty-four (94%) of the patients achieved at least 3 million CD34 cells/kg within 2 days of apheresis. The median CD34 cells/kg after 1 day of collection was 3.9 million (range; 0.7 to 9.2) and after two days of collection was 7.02 million (range: 1.1–16.5). Two patients failed the mobilization (<2.5 million CD34 cells/kg). There were no grade 3 or 4 non-hematological adverse events and one patient experienced grade 4 thrombocytopenia. The most common grade 1 or 2 adverse events seen were gastrointestinal, namely nausea, diarrhea and abdominal pain or bloating. Grade 1 dizziness was reported in 8 patients. Overall, the infusion was well tolerated. Conclusion:Intravenously administered plerixafor is an effective strategy for mobilization in this patient group with low rate of failure to mobilize. It is well tolerated with toxicity comparable to the SQ administration. It also offers flexibility in patient scheduling with a convenient schedule for early morning infusion followed by apheresis later in the day. Disclosures:Kumar:Merck: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc.: Research Funding; Genzyme: Research Funding; Novartis: Research Funding. Off Label Use: Use of the investigational agent MLN9708 for the treatment of previously untreated multiple myeloma. Lacy:Celgene: Research Funding.

Strategies to Prevent or Reverse Immune Responses Against Factor In Gene Replacement for Hemophilia B.

AbstractAbstract 3761Factor IX (F.IX) deficiency resulting from large gene deletions or other null mutations can have severe ramifications due to the lack of F.IX immune recognition as a self-protein in hemophilia B. Hence, therapeutic treatment with F.IX protein can result in inhibitory antibody formation, thereby negating therapy. Muscle-directed gene therapy is an attractive novel treatment modality because of the safety and simplicity of vector administration but is hampered by an increased risk of such immune responses. Previously, we demonstrated that the drug cocktail of immune suppressants Rapamycin (Rapa), IL-10 and a specific peptide (encoding a dominant CD4+ T cell epitope) caused an induction of Treg with a concomitant antigen specific apoptosis of Teff (J. Thromb. Haemost. 7:1523, 2009). This protocol was effective in preventing inhibitor formation against human F.IX upon subsequent IM administration of an AAV-hF.IX vector in C3H/HeJ hemophilia B mice with targeted F.IX gene deletion. Here, we used this model to test whether an inhibitor response to gene therapy can be reversed. IM injection of the AAV1-hF.IX vector resulted in a high-titer inhibitory antibody response of on average 8 BU (8 mg IgG1/ml plasma) within 1 month. Transduced mice had aPTTs of ≂f80 sec and lacked systemic hFIX expression. Subsequently, the animals (n=5) were treated with IP injections of rapa/IL-10/FIX peptide 3 times/week for 4 weeks, resulting in a significant reduction of inhibitor titers to <2 BU by 1 month after tolerization. Unresponsiveness was maintained for >5 months. This reduction in the inhibitors was accompanied by a drastic decrease in IgG1 response and an increase in hF.IX production from 0 ng/ml before tolerization to a stable level of 200–650 ng/ml. Clotting times gradually declined to 58–62sec, representing a coagulation activity of ≂f1% of normal. In contrast, transduced control mice that were not treated with the tolerance protocol failed to show hF.IX expression or correction of aPTTs and maintained inhibitors for the duration for the experiment. A problem with prophylactic use of this protocol against inhibitor formation is that the specific T cell epitopes may not be known in a patient. However, when we used hF.IX protein instead of the peptide, the tolerance protocol was not effective and thus required further optimization. In humans, rapamycin is mostly given in oral form. We compared IP, SQ, IV, and oral routes of drug administration in a defined T cell receptor transgenic model (DO.11.10 Rag-/- mice). Induction of ovalbumin-specific CD4+CD25+FoxP3+ Treg up to 20% of CD4+ T cells was achieved with all these routes, except that oral administration of rapa was less effective. We reasoned that more frequent oral rapa administration may overcome this limitation. After additional optimization steps, we tested the following protocol. Rapa was given orally on a daily schedule for 1 month to the hemophilia B mice, during which time a subtherapeutic dose of hF.IX protein (0.1 IU) was given IV twice/week. Vector was given IM 1 week before the end of the tolerance protocol. This regiment was effective and prevented inhibitor formation (<1 BU), resulting in systemic hF.IX expression. We propose that alternative protocols based on use of rapamyin and either the entire cognate protein or a known T cell epitope (which can be mapped during the course of an immune response) can applied to prevent or reverse inhibitor responses in gene therapy for hemophilia. Disclosures:Herzog:Genzyme Corp: Patents & Royalties.

A Phase I Biological Study of Azacitidine (Vidaza) to Determine the Optimal Biological Dose and Route of Administration.

Abstract 1753Poster Board I-779 BackgroundAzacitidine (5-azacytidine, Vidaza) is a DNA methylation inhibitor with used to treat myelodysplastic syndrome (MDS). The studies which led to FDA approval based dosing and administration guidelines on clinical response. In vitro studies have demonstrated that azacitidine exerts its effect by inhibiting DNA methyltransferase in hypermethylated tumor suppressor genes in malignant cells. Research to date has not linked azacitidine dosing with biochemical and clinical response in vivo. The degree of DNA repetitive element sequence methylation (such as LINE-1) has been demonstrated to correlate with global DNA methylation and may be used to determine DNA methylation changes after treatment with azacitidine. We have conducted a phase I study to link clinical and biologic response to azacitidine. This study aims to determine the optimal dose and route of administration for azacitidine to inhibit global DNA methylation levels in the peripheral blood of patients with hematologic malignancies. MethodsPatients with hematologic malignancy who provided informed consent were eligible for study inclusion, with enrollment criteria based on the specific malignancy. Patients were enrolled into one of five dose level treatment groups (25mg, 50mg, 75mg, 100mg or 150mg IV per m2 per day for 5 days) for the first course of therapy. On day 28, all patients received a course of 75mg/m2/day IV for 5 days. Subcutaneous dosing of 75mg/m2/day for 5 days was used for course three. Patients received 75mg/m2/day either SQ or IV x 5 days every 4 weeks for course four and beyond. Peripheral blood was collected on days 1, 3, and 5 during each course, and global DNA methylation was measured using bisulfite-PCR Pyrosequencing of the 6 DNA repetitive elements (LINE1, AluYb8, AluSq, Sat-alpha, D4Z4, NBL-2). Additionally, gene promoter specific DNA methylation was assessed in a subset of patients using the Illumina GoldenGate Bead Array DNA Methylation Assay which measures DNA methylation of 1505 CpG sites (807 genes). ResultsSeventeen patients were treated (3 at 25mg, 4 at 50mg, 4 at 75mg, 3 at 100mg, and 3 at 150mg/m2). Diagnosis included 5 patients with MDS, 10 patients with AML (2 untreated older patients, 7 relapsed or refractory patients), 1 patient with CML (Imatinib refractory), and 1 patient with non-Hodgkin's lymphoma (relapsed disease). At the time of submission, 14 patients were evaluable for response with 4 CR (1 mCr, 1 CRp), 1 PR, 6 SD and 3 PD reported. The median number of cycles given was 3 (range 1-14+). LINE1 DNA methylation decreased by 1.4, 2.3, 4.8, 1.9 and 4.0% on day 5 for the 25mg, 50mg, 75mg, 100mg, and 150mg/m2 course one dose levels respectively. Mean decrease in LINE1 DNA methylation with 75mg/m2 IV was 3.7% and only 2.6% by 75mg/m2 of azacitidine SQ. There was a large amount of inter-patient variability but less intra-patient variability in DNA methylation response to azacitidine. ConclusionAzacitidine is effective at inhibiting DNA methylation at multiple dose levels for both IV and SQ routes of administration. There is a high degree of patient-to-patient variability in DNA methylation changes, although 75mg/m2 lead to the greatest mean decrease in DNA methylation by a 5 day IV regimen. Measurement of DNA methylation of LINE1 and AluYb8 repetitive elements were the best surrogate markers for measuring overall changes in gene specific promoter DNA methylation when compared with 807 genes assessed by the Illumina GoldenGate platform. High-throughput gene promoter DNA methylation analysis revealed subtle changes in DNA methylation, though gene specific changes could not be linked to therapeutic activity. DisclosuresOff Label Use: Azacitidine in hematologic malignancies other than MDS. Mohrbacher:Celgene: Honoraria, Speakers Bureau. Gorospe:Novartis: Honoraria, Speakers Bureau. Yang:Celgene: Honoraria, Research Funding, Speakers Bureau.

Phasee I Biological Study of IV and SQ Azacitidine (Vidaza®) to Determine the Optimal Biological Dose in Hematologic Malignancies

Azacitidine (5-azacytidine, Vidaza) is a DNA methylation inhibitor that has been shown to have a survival benefit in patients with high-risk myelodysplastic syndrome and is under investigation in other cancers. The purpose of this study was to determine the optimal dose and route of administration for azacitidine to inhibit DNA methylation in patients with hematologic malignancies. Eligibility included patients with hematologic malignancy who provided informed consent. Enrollment criteria varied depending on the type of cancer. Patients were enrolled into 5 escalating dose levels for the first course of therapy (25mg, 50mg, 75mg, 100mg or 150mg IV per m2 per day for 5 days). On day 28 all patients received 75mg/m2/day IV for 5 days. For course 3 patients received 75mg/m2/day SQ × 5 days. Beyond course 3 patients received 75mg/m2/day either SQ or IV × 5 days every 4 weeks. Peripheral blood was collected on days 1, 3 and 5 for each course and LINE1 DNA methylation was measured using bisulfite-PCR Pyrosequencing to measure global DNA methylation changes induced by azacitidine. To date, 17 patients have been treated (3 at 25mg, 4 at 50mg, 4 at 75mg, 3 at 100mg, and 3 at 150mg/m2). Diagnosis included 6 patients with MDS, 9 patients with AML (3 untreated older patients, 6 relapsed or refractory patients), 1 patient with CML (imatinib refactory), and 1 patient with non-Hodgkin's lymphoma (relapsed disease). At the time of submission (August 2008) 12 patients were evaluable for response with 1 CR, 3 PR, 7 SD and 1 PD reported. The median number of cycles given was 2 (range 0–12+). LINE1 DNA methylation decreased by 1.4, 2.3, 4.8, 1.9 and 4.0% on day 5 for the 25mg, 50mg, 75mg, 100mg, and 150mg/m2 dose levels respectively. Mean decrease in LINE1 DNA methylation with 75mg/m2 IV was 3.7% and only 2.6% by 75mg/m2 of azacitidine SQ. There was a large amount of inter-patient variability but less intra-patient variability in DNA methylation response to azacitidine. DNA methylation and data analysis is ongoing. Azacitidine is effective at inhibiting DNA methylation at multiple dose levels for both IV and SQ routes of administration. There is a high degree of patient-to-patient variability in DNA methylation changes, but 75mg/m2 lead to the greatest mean decrease in DNA methylation by a 5 day IV regimen. Updated analysis for this study will be presented.