Safety evaluation of orally administered afoxolaner and milbemycin oxime in eight-week-old dogs.

The safety profile of afoxolaner (an isoxazoline molecule) when combined with milbemycin oxime (a macrocyclic lactone) was evaluated according to the regulatory requirements when administered six times orally in a soft chewable formulation at a dose of at least 1×, 3×, or 5× the maximum exposure dose in 8-week-old Beagle dogs. Thirty-two healthy puppies (16 males and 16 females) were enrolled and allocated randomly to one of four treatment groups. Three doses were administered at 28-day intervals (Days 0, 28, and 56), followed by three additional doses administered with 14-day intervals (Days 84, 98, and 112). The study ended on Day 126. Treatment groups were as follows: Group 1: untreated, sham-dosed control; Group 2: afoxolaner/milbemycin oxime chews administered at a dose of at least 5 and 1 mg/kg, respectively (1×); Group 3: afoxolaner/milbemycin oxime chews administered at a dose of at least 15 and 3 mg/kg, respectively (3); and Group 4: afoxolaner/milbemycin oxime chews administered at a dose of at least 25 and 5 mg/kg, respectively (5×). All dogs were examined for general health twice a day beginning on Day -14. Physical examinations, and blood collections for clinical pathology analysis and afoxolaner and milbemycin oxime plasma concentrations, were performed throughout the study. No afoxolaner/milbemycin oxime treatment-related changes were observed in growth, physical variables, clinical pathology variables, or tissues examined histologically. No clinically relevant or statistically significant health abnormalities related to the administration of afoxolaner/milbemycin oxime were observed. No signs of macrocyclic lactone sensitivity were observed at any time during the study. Vomiting and diarrhea were observed sporadically across all groups including the controls. Based upon the results of this study, afoxolaner/milbemycin oxime soft chewables were shown to be safe when administered repeatedly at up to 5× the maximum exposure dose in dogs as young as 8 weeks of age.

The intravenous and oral pharmacokinetics of afoxolaner and milbemycin oxime when used as a combination chewable parasiticide for dogs.

The pharmacokinetics of afoxolaner and milbemycin oxime (A3 and A4 forms) in dogs were evaluated following the oral administration of NexGard Spectra® (Merial), a fixed combination chewable formulation of these two active pharmaceutical ingredients. Absorption of actives was rapid at levels that provide the minimum effective doses of 2.5 mg/kg and 0.5 mg/kg of afoxolaner and milbemycin oxime, respectively. The time to maximum afoxolaner plasma concentrations (tmax ) was 2-4 h. The milbemycin tmax was 1-2 h. The terminal plasma half-life (t1/2 ) and the oral bioavailability were 14 ± 3 days and 88.3% for afoxolaner, 1.6 ± 0.4 days and 80.5% for milbemycin oxime A3 and 3.3 ± 1.4 days and 65.1% for milbemycin oxime A4. The volume of distribution (Vd ) and systemic clearance (Cls) were determined following an IV dose of afoxolaner or milbemycin oxime. The Vd was 2.6 ± 0.6, 2.7 ± 0.4 and 2.6 ± 0.6 L/kg for afoxolaner, milbemycin oxime A3 and milbemycin oxime A4, respectively. The Cls was 5.0 ± 1.2, 75 ± 22 and 41 ± 12 mL/h/kg for afoxolaner, milbemycin oxime A3 and milbemycin oxime A4, respectively. The pharmacokinetic profile for the combination of afoxolaner and milbemycin oxime supports the rapid onset and a sustained efficacy for afoxolaner against ectoparasites and the known endoparasitic activity of milbemycin oxime.

Pharmacokinetics of gamithromycin in cattle with comparison of plasma and lung tissue concentrations and plasma antibacterial activity.

The pharmacokinetics (PK) and dose proportionality of gamithromycin (ZACTRAN), a novel azalide, after a single intravenous (i.v.) dose of 3 mg/kg or subcutaneous (s.c.) injection at 3, 6 and 9 mg/kg body weight were studied in 13 male castrate and 13 female Angus cattle. Following i.v. administration, the mean area under the curve extrapolated to infinity (AUC(inf)) was 4.28 +/- 0.536 microgxh/mL, and mean elimination half-life (t(1/2)) was 44.9 +/- 4.67 h, with a large volume of distribution (V(ss)) of 24.9 +/- 2.99 L/kg and a high clearance rate (Cl(obs)) of 712 +/- 95.7 mL/h/kg. For cattle treated with s.c. injection of 3, 6 or 9 mg/kg, mean AUC(inf) values were 4.55 +/- 0.690, 9.42 +/- 1.11 and 12.2 +/- 1.13 microgxh/mL, respectively, and the mean elimination half-lives (t(1/2)) were 51.2 +/- 6.10, 50.8 +/- 3.80 and 58.5 +/- 5.50 h. Gamithromycin was well absorbed and fully bioavailable (97.6-112%) after s.c. administration. No statistically significant (alpha = 0.05) gender differences in the AUC(Inf) or elimination half-life values were observed. Dose proportionality was established based on AUC(Inf) over the range of 0.5 to 1.5 times of the recommended dosage of 6 mg/kg of body weight. Further investigations were conducted to assess plasma PK, lung/plasma concentration ratios and plasma antibacterial activity using 36 cattle. The average maximum gamithromycin concentration measured in whole lung homogenate was 18 500 ng/g at first sampling time of 1 day ( approximately 24 h) after treatment. The ratios of lung to plasma concentration were 265, 410, 329 and 247 at 1, 5, 10 and 15 days postdose. The lung AUC(inf) was 194 times higher than the corresponding plasma AUC(inf). The apparent elimination half-life for gamithromycin in lung was 90.4 h ( approximately 4 days). Antibacterial activity was observed with plasma collected at 6 h postdose with a corresponding average gamithromycin plasma concentration of 261 ng/mL. In vitro plasma protein binding in bovine plasma was determined to be 26.0 +/- 0.60% bound over a range of 0.1-3.0 microg/mL of gamithromycin. The dose proportionality of AUC, high bioavailability, rapid and extensive distribution to lung tissue and low level of plasma protein binding are beneficial PK parameters for an antimicrobial drug used for the treatment and prevention of bovine respiratory disease.

Testing the safety of clinical-grade mature autologous myeloid DC in a phase I clinical immunotherapy trial of CML.

BACKGROUND: We conducted a phase I clinical immunotherapy trial of CML to evaluate the safety of a clinical-grade leukemic DC product standardized for purity and mature phenotype. METHODS: We injected autologous DC into patients in late chronic or accelerated phases of CML. The patients received mature CD83+ and bcr-abl+ DC prepared from CD14+ cells. Two cohorts of three patients received four injections each of 3 x 10(6) DC and 15 x 10(6) DC/injection, respectively. The first patient was studied before imatinib mesylate (IM) was available, four patients were treated concurrently with IM therapy and one did not tolerate the IM and was off the drug at the time of DC therapy. IM effects on WBC counts precluded DC preparation in numbers sufficient for further dose escalation. The first patient received DC s.c. and all subsequent patients received DC into a cervical lymph node under ultrasound guidance. RESULTS: DC injections were well tolerated. We observed no clinical responses. T cells drawn later in the course of therapy were more sensitive to stimulation by CML DC in vitro. DISCUSSION: The increase in T-cell sensitivity to CML-specific stimulation that accompanied active immunization by CML DC justifies further clinical studies, possibly with modifications such as an increased frequency and number of DC injections.

Allogeneic stem cell transplantation and donor lymphocyte infusions for chronic myelomonocytic leukemia.

Prognosis in chronic myelomonocytic leukemia (CMML) is unfavorable and the optimal therapy remains uncertain. Currently, allogeneic stem cell transplantation is the only known curative therapeutic option. However, the data available are limited and restricted to small retrospective series. There is even less information on the use of donor lymphocyte infusions (DLI) for this disease. We reviewed our experience of allogeneic stem cell transplantation and DLI for adults with CMML. Seventeen consecutive adults underwent allogeneic stem cell transplantation from related (n=14) or unrelated (n=3) donors. Median age was 50 years (range 26-60). Seven patients (41%) demonstrated relapse or persistent disease at a median of 6 months (range 3-55.5). Five patients underwent DLI for morphologic relapse and one for mixed donor chimerism. Two patients achieved durable complete remissions of 15 months each. The overall transplant-related mortality was 41% (n=7). With a median follow-up of 34.5 months, three patients (18%) currently remain alive and in continuous CR. The current study demonstrates a graft-versus-leukemia effect in CMML, both for allogeneic stem cell transplantation and for DLI. Nevertheless, consistent with reported experience of others, overall outcomes remain less than optimal and unpredictable.

Absolute lymphocyte count recovery after induction chemotherapy predicts superior survival in acute myelogenous leukemia.

Absolute lymphocyte count (ALC) recovery postautologous stem cell transplantation is an independent predictor for survival in acute myelogenous leukemia (AML). The role of ALC recovery after induction chemotherapy (IC) in AML is unknown. We hypothesize that ALC recovery after IC has a direct impact on survival. We have now evaluated the impact of ALC recovery after IC on overall survival (OS) and leukemia-free survival (LFS) in 103 consecutive, newly diagnosed AML patients treated with standard IC and consolidation chemotherapy (CC) from 1998 to 2002. ALC recovery was studied at days 15 (ALC-15), 21 (ALC-21), 28 (ALC-28) after IC and before the first CC (ALC-CC). Superior OS and LFS at each time point were observed with an ALC-15, ALC-21, ALC-28, and ALC-CC > or = 500 cells/microl. Patients with an ALC > or = 500 cells/microl at all time points vs those who did not have superior OS and LFS (not reached vs 13 months, P<0.0001; and not reached vs 11 months, P<0.0001, respectively). Multivariate analysis demonstrated ALC > or = 500 cells/microl at all time points to be an independent prognostic factor for survival. Our data suggest a critical role of lymphocyte (immune) recovery on survival after IC in AML.

Early lymphocyte recovery is a predictive factor for prolonged survival after autologous hematopoietic stem cell transplantation for acute myelogenous leukemia.

Absolute lymphocyte count (ALC) recovery correlates with survival after autologous hematopoietic stem cell transplantation (AHSCT) for patients with multiple myeloma, non-Hodgkin's lymphoma, and metastatic breast cancer. The role of ALC recovery in relationship to clinical outcome after AHSCT in patients with acute myelogenous leukemia is unknown. We analyzed 45 patients who underwent AHSCT at Mayo Clinic, Rochester, Minnesota between 1990 and 2000. The ALC threshold was selected at 500 cells/microl on day 15 post-AHSCT based on our previous studies. Thirty-two females and 13 males were included in the study with a median age of 45 years (range 12-75). The median follow-up was 14 months with a maximum of 129 months. The median overall and leukemia-free survival were significantly better for the 23 patients with ALC at day 15 > or =500 cells/microl compared with 22 patients with ALC <500 cells/microl (not yet reached vs 10 months, P < 0.0009; 105 vs 9 months, P < 0.0008, respectively). In conclusion, ALC > or =500 cells/microl on day 15 post-AHSCT is associated with better survival in acute myelogenous leukemia and requires further studies.

Acute leukemia during pregnancy: a single institutional experience with 17 cases.

We reviewed the medical records of 17 consecutive patients with concomitant acute leukemia and pregnancy seen at our institution over a 37-year period. Fifteen cases each were either newly diagnosed or classified as acute myeloid leukemia (AML). Seven diagnoses (41%) occurred in the first, 7 (41%) in the second, and 3 (18%) in the third trimester. In general, nine patients received chemotherapy while pregnant-eight in the second trimester and one in the third. The overall complete remission rate among the 13 patients with newly diagnosed AML was 69%, compared with 86% in those who were pregnant during chemotherapy. Long-term survival was documented in five of the nine complete responders. Three of four patients who elected to delay treatment until after delivery died within days of starting chemotherapy. Unintentional fetal loss occurred in four patients (29%), including two without exposure to chemotherapy. There were no instances of congenital malformation. The results from the current study confirm that pregnancy per se may not affect the outcome of chemotherapy in AML. In addition, it is suggested that treatment delays may compromise maternal outcome without improving pregnancy outcome.

Amifostine alone and in combination with erythropoietin for the treatment of favorable myelodysplastic syndrome.

Ten anemic patients with favorable myelodysplastic syndrome (MDS) were first treated with two 5-week courses of amifostine alone (each course consisted of 200 mg/m(2) of the drug given intravenously three times a week for 3 weeks), followed by an additional two courses combined with subcutaneous erythropoietin (EPO) (150 U/kg, three times a week for 8 weeks). The study patients either had previously failed to respond to treatment with EPO or had pretreatment serum EPO levels of more than 100 mU/ml. None of the patients experienced a complete or partial response in anemia or other cytopenias. We conclude that amifostine alone or in combination with EPO has limited therapeutic activity in MDS.

Development of hepatic veno-occlusive disease after Mylotarg infusion for relapsed acute myeloid leukemia.

Mylotarg (gemtuzumab zogamicin) is a conjugated monoclonal antibody that has recently become available for use in patients with relapsing or refractory acute myeloid leukemia. Reversible hepatotoxicity is common after administration. We describe the first report of hepatic veno-occlusive disease (HVOD) developing after Mylotarg infusion in a patient who underwent hematopoietic stem cell transplantation 8 months earlier. Certain antineoplastic agents have been implicated as a cause of HVOD, but the disease is most commonly seen within 30 days after hematopoietic stem cell transplantation. The possible association between Mylotarg infusion and HVOD is discussed.

Low-dose cytarabine maintenance therapy vs observation after remission induction in advanced acute myeloid leukemia: an Eastern Cooperative Oncology Group Trial (E5483).

The Eastern Cooperative Oncology Group (ECOG) conducted a prospective phase III study in patients with relapsed/refractory acute myeloid leukemia (AML) to evaluate whether administration of repeated courses of low-dose cytarabine (LDAC) maintenance therapy after induction of complete remission in advanced AML would improve disease-free and overall survival. Patients with AML in second/later relapse or refractory disease were first treated with a combination of high-dose cytarabine and amsacrine. Those who achieved complete remission were then randomized to observation or to receive LDAC, 10 mg/m2 subcutaneously twice a day x2 21 days every 2 months until relapse occurred. Of 86 patients eligible for randomization, 41 patients were assigned to receive LDAC and 45 patients to observation. The median disease-free survival was 7.4 months for patients assigned to LDAC compared to 3.3 months for patients receiving no additional therapy, P= 0.084. The median survival from randomization was 10.9 months and 7.0 months for patients receiving LDAC maintenance chemotherapy and observation, respectively (P= 0.615). The data from this study suggest that LDAC maintenance therapy given to patients with advanced AML who achieve complete remission can increase disease-free survival compared to observation, but does not improve overall survival. Nevertheless, because of the ineffectiveness and toxicity of intensive post-remission chemotherapy in this circumstance, LDAC maintenance therapy, a tolerable outpatient regimen, offers the potential for improved quality of life.

All-trans-retinoic acid-induced myositis: a description of two patients.

All-trans-retinoic acid (ATRA) induces complete clinical remissions in a high proportion of patients with acute promyelocytic leukemia and has become the standard induction therapy. Its use as a single agent results in short-lived remissions; thus, cytotoxic drugs are used for "consolidation" therapy. Side effects reported during treatment with ATRA include retinoic acid syndrome and Sweet's syndrome. Sweet's syndrome has been associated with acute myelogenous leukemia at presentation, but only two cases of Sweet's syndrome involving the musculoskeletal system in patients treated with ATRA have been described. We describe two additional patients with acute promyelocytic leukemia who had unexplained fever and myalgias (cutaneous lesions in one patient) during induction therapy with ATRA. Radiologic findings were similar to those in previously reported ATRA-associated Sweet's syndrome of the musculoskeletal system. The clinical course was characterized by a rapid resolution of the symptoms during treatment with dexamethasone. Recognition of the syndrome is important, especially considering the rapid resolution of symptoms after early institution of therapy with corticosteroids.

Treatment of unfavorable myelodysplastic syndrome with all-trans-retinoic acid and subcutaneous Ara C.

Thirty patients with high-risk myelodysplastic syndrome, defined as > 5% bone marrow blasts, were treated with a combination of oral all-trans-retinoic acid (45 mg/m2 daily) and subcutaneous AraC (10 mg/m2) on days 1-14 of each 28-35 day cycle repeated for 2-6 cycles. Complete remission lasting 9, 12, and 15 months was achieved in three patients. Partial and minor response did not translate into meaningful clinical improvement, like complete responders. Overall incidence of leukemia transformation and survival of this cohort of patients was no different from the expected outcome for a group of patients with similar characteristics.

Chronic lymphocytic leukaemia with symptomatic diffuse central nervous system infiltration responding to therapy with systemic fludarabine.

B-cell chronic lymphocytic leukaemia is an indolent disease characterized by the insidious accumulation of small mature-appearing lymphocytes in the peripheral blood, bone marrow and lymphoid tissues. Direct symptomatic invasion of the central nervous system is exceedingly rare and, to our knowledge, only three cases histologically confirmed as true chronic lymphocytic leukaemia have been reported in the literature. We describe the first case of early Rai stage B-cell chronic lymphocytic leukaemia presenting with symptomatic infiltration of the brain and spinal cord which could be demonstrated radiographically by magnetic resonance imaging. The diagnosis was confirmed by examination of peripheral blood, cerebrospinal fluid, brain and bone marrow biopsies, both morphologically and immunophenotypically by means of flow cytometric analysis. The patient demonstrated a complete response to therapy with standard-dose systemic fludarabine and remains in complete remission 6 months after completion of therapy.

Multiple lymphoid nodules in bone marrow have the same clonality as underlying myelodysplastic syndrome recognized with fluorescent in situ hybridization technique.

Benign nodular lymphoid lesions are not rare in the bone marrow of patients with myelodysplastic syndrome (MDS). Herein, we report a case of MDS with clonal lymphoid aggregates in the bone marrow but without evidence of systemic lymphoma. The case of a 71-year-old man was evaluated for cytopenia. His bone marrow was initially hypocellular, with 10% blasts and a few small lymphoid aggregates. The diagnosis of refractory anemia with excess blasts was made. The disease progressed gradually, and he received erythropoietin and granulocyte colony-stimulating factor for a short time. Forty-two months later, acute leukemia (M1) developed, with 60% to 70% blasts in the bone marrow. The bone marrow also showed large aggregates of lymphocytes. Immunohistochemical study of these cells in the nodular lesions showed 50% CD3+ and 50% CD20+. Cytogenetic and molecular genetic studies revealed monosomy 7 and T- and B-cell clonal gene rearrangement. Fluorescent in situ hybridization study with centromere-specific probes of a bone marrow specimen showed monosomy 7 in both nodular lymphoid lesions and surrounding bone marrow cells, indicating that both processes originated from the same abnormal pluripotential progenitor.

Nodular lesions of monocytic component in myelodysplastic syndrome.

We report 4 unusual cases of myelodysplastic syndrome with distinct persistent nodular lesions noted on serial bone marrow examinations, even during remission. The lesions were predominantly composed of immature monocytes that stained positively for CD68. Trisomy 9 and 11 were demonstrated in the cells of the nodular lesions and surrounding marrow of 1 patient, indicating the same clonal origin. Evaluation of p53 glycoprotein, retinoblastoma protein (pRb), proliferation-related protein (Ki-67), multiple drug-resistant enzyme glutathione-S-transferase pi, and topoisomerase IIalpha (Topo IIalpha) revealed decreased topoisomerase expression within the nodular lesions compared with the surrounding marrow and absence of Ki-67 antigen within nodular lesions. Most cells in the lesion were not in a proliferative cycle, with very low expression of Topo IIalpha, which may explain the apparent drug resistance of these nodular lesions.