Luke A. Wittenburg

Phase I clinical trial and pharmacodynamic evaluation of combination hydroxychloroquine and doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma

Autophagy is a lysosomal degradation process that may act as a mechanism of survival in a variety of cancers. While pharmacologic inhibition of autophagy with hydroxychloroquine (HCQ) is currently being explored in human clinical trials, it has never been evaluated in canine cancers. Non-Hodgkin lymphoma (NHL) is one of the most prevalent tumor types in dogs and has similar pathogenesis and response to treatment as human NHL. Clinical trials in canine patients are conducted in the same way as in human patients, thus, to determine a maximum dose of HCQ that can be combined with a standard chemotherapy, a Phase I, single arm, dose escalation trial was conducted in dogs with spontaneous NHL presenting as patients to an academic, tertiary-care veterinary teaching hospital. HCQ was administered daily by mouth throughout the trial, beginning 72 h prior to doxorubicin (DOX), which was given intravenously on a 21-d cycle. Peripheral blood mononuclear cells and biopsies were collected before and 3 d after HCQ treatment and assessed for autophagy inhibition and HCQ concentration. A total of 30 patients were enrolled in the trial. HCQ alone was well tolerated with only mild lethargy and gastrointestinal-related adverse events. The overall response rate (ORR) for dogs with lymphoma was 93.3%, with median progression-free interval (PFI) of 5 mo. Pharmacokinetic analysis revealed a 100-fold increase in HCQ in tumors compared with plasma. There was a trend that supported therapy-induced increase in LC3-II (the cleaved and lipidated form of microtubule-associated protein 1 light chain 3/LC3, which serves as a maker for autophagosomes) and SQSTM1/p62 (sequestosome 1) after treatment. The superior ORR and comparable PFI to single-agent DOX provide strong support for further evaluation via randomized, placebo-controlled trials in canine and human NHL.

The histone deacetylase inhibitor valproic acid sensitizes human and canine osteosarcoma to doxorubicin

PurposeOsteosarcoma (OS) remains an incurable and ultimately fatal disease in many patients, and novel forms of therapy are needed. Improved models of OS that more closely mimic human disease would provide more robust information regarding the utility of novel therapies. Spontaneous OS in dogs may provide such a model. Pharmacologic inhibition of histone deacetylase (HDAC) enzymes has a variety of anti-tumor effects but may demonstrate the most utility when utilized in combination with standard cytotoxic therapies. We sought to determine the in vitro and in vivo effects of the HDAC inhibitor valproic acid (VPA) on doxorubicin (DOX) sensitivity in canine and human OS.MethodsWe evaluated the in vitro anti-proliferative and apoptotic effects of VPA/DOX combination treatment, alterations in histone acetylation and nuclear DOX accumulation resulting from VPA treatment, and the in vivo efficacy of combination therapy in a xenograft model.ResultsTreatment of canine and human OS cell lines with clinically achievable VPA concentrations resulted in increased histone acetylation but modest anti-proliferative effects. Pre-incubation with VPA followed by doxorubicin (DOX) resulted in significant growth inhibition and potentiation of apoptosis, associated with a dose-dependent increase in nuclear DOX accumulation. The combination of VPA and DOX was superior to either monotherapy in a canine OS xenograft model.ConclusionThese results demonstrate a rationale for the addition of HDAC inhibitors to current protocols for the treatment of OS and illustrate the similarities in response to HDAC inhibitors between human and canine OS, lending further credibility to the canine OS model.

Phase I Pharmacokinetic and Pharmacodynamic Evaluation of Combined Valproic Acid/Doxorubicin Treatment in Dogs with Spontaneous Cancer

Purpose: Histone deacetylase inhibitors (HDACi) are targeted anticancer agents with a well-documented ability to act synergistically with cytotoxic agents. We recently showed that the HDACi valproic acid sensitizes osteosarcoma cells to doxorubicin in vitro and in vivo. As there are no published reports on the clinical utility of HDACi in dogs with spontaneous cancers, we sought to determine a safe and biologically effective dose of valproic acid administered prior to a standard dose of doxorubicin. Methods: Twenty-one dogs were enrolled into eight cohorts in an accelerated dose-escalation trial consisting of pretreatment with oral valproic acid followed by doxorubicin on a three-week cycle. Blood and tumor tissue were collected for determination of serum valproic acid concentration and evaluation of pharmacodynamic effects by immunofluorescence cytochemistry and immunohistochemistry. Serum and complete blood counts were obtained for determination of changes in doxorubicin pharmacokinetics or hematologic effects. Results: All doses of valproic acid were well tolerated. Serum valproic acid concentrations increased linearly with dose. Doxorubicin pharmacokinetics were comparable with those in dogs receiving doxorubicin alone. A positive correlation was detected between valproic acid dose and histone hyperacetylation in peripheral blood mononuclear cells. No potentiation of doxorubicin-induced myelosuppression was observed. Histone hyperacetylation was documented in tumor and peripheral blood mononuclear cells. Responses included 2 of 21 complete, 3 of 21 partial, 5 of 21 stable disease, and 11 of 21 progressive disease. Conclusions: Valproic acid can be administered to dogs at doses up to 240 mg/kg/day prior to a standard dose of doxorubicin. In addition, we have developed the pharmacokinetic/pharmacodynamic tools necessary for future studies of novel HDACi in the clinical setting of canine cancer. Clin Cancer Res; 16(19); 4832–42. ©2010 AACR.

A Systems Biology Approach to Identify Molecular Pathways Altered by HDAC Inhibition in Osteosarcoma

Osteosarcoma (OS) is the most common primary tumor in humans and dogs affecting the skeleton, and spontaneously occurring OS in dogs serves as an extremely useful model. Unacceptable toxicities using current treatment protocols prevent further dose‐intensification from being a viable option to improve patient survival and thus, novel treatment strategies must be developed. Histone deacetylase inhibitors (HDACi) have recently emerged as a promising class of therapeutics demonstrating an ability to enhance the anti‐tumor activity of traditional chemotherapeutics. To date, gene expression analysis of OS cell lines treated with HDACi has not been reported, and evaluation of the resultant gene expression changes may provide insight into the mechanisms that lead to success of HDACi. Canine OS cells, treated with a clinically relevant concentration of the HDACi valproic acid (VPA), were used for expression analysis on the Affymetrix canine v2.0 genechip. Differentially expressed genes were grouped into pathways based upon functional annotation; pathway analysis was performed with MetaCore and Ingenuity Pathways Analysis software. Validation of microarray results was performed by a combination of qRT‐PCR and functional/biochemical assays revealing oxidative phosphorylation, cytoskeleton remodeling, cell cycle, and ubiquitin‐proteasome among those pathways most affected by HDACi. The mitomycin C‐bioactivating enzyme NQ01 also demonstrated upregulation following VPA treatment, leading to synergistic reductions in cell viability. These results provide a better understanding of the mechanisms by which HDACi exert their effect in OS, and have the potential to identify biomarkers that may serve as novel targets and/or predictors of response to HDACi‐containing combination therapies in OS. J. Cell. Biochem. 113: 773–783, 2012.

Optimizing preclinical study design in oncology research.

The current drug development pathway in oncology research has led to a large attrition rate for new drugs, in part due to a general lack of appropriate preclinical studies that are capable of accurately predicting efficacy and/or toxicity in the target population. Because of an obvious need for novel therapeutics in many types of cancer, new compounds are being investigated in human Phase I and Phase II clinical trials before a complete understanding of their toxicity and efficacy profiles is obtained. In fact, for newer targeted molecular agents that are often cytostatic in nature, the conventional preclinical evaluation used for traditional cytotoxic chemotherapies utilizing primary tumor shrinkage as an endpoint may not be appropriate. By utilizing an integrated pharmacokinetic/pharmacodynamic approach, along with proper selection of a model system, the drug development process in oncology research may be improved leading to a better understanding of the determinants of efficacy and toxicity, and ultimately fewer drugs that fail once they reach human clinical trials.

Effect of topical naltrexone 0.3% on corneal sensitivity and tear parameters in normal brachycephalic dogs

OBJECTIVE To determine the effect of topical naltrexone 0.3% on tear production, corneal sensitivity, and tear film stability in normal brachycephalic dogs. ANIMALS STUDIED Twenty-two normal brachycephalic dogs. PROCEDURES Measurements of tear production (Schirmer tear test I and II), intraocular pressure (IOP), central corneal sensitivity (CS), and tear film breakup time (TFBUT) were collected at time 0, 1, and 24 h after administration of either naltrexone (NTX) 0.3% or placebo (SV). Naltrexone or SV was then administered once daily for 1 week, and the above measurements were repeated at 7 days, then again 7 days after discontinuing medication. Owners scored the degree of comfort, redness, rubbing, squinting, and tearing. Serum was collected at time 0, 1, 24 h, and 7 days to determine systemic concentrations. RESULTS Owners reported no significant change in the degree of comfort, redness, rubbing, squinting, or tearing. Naltrexone was detected in serum of all treated dogs 1-h postadministration (average: 908 pg/mL, range: 319-1570 pg/mL) and in two dogs at the 1-week time point. Naltrexone was not detected at the 24-h time point. There was no significant effect of NTX on STT1, STT2, IOP, CS, or TFBUT. CONCLUSIONS Naltrexone 0.3% is well tolerated and safe when applied topically to the eye once daily. Naltrexone 0.3% did not show any significant effects on corneal parameters as measured in this study. At once, daily dosing NTX is systemically absorbed; however, the degree of systemic absorption is not likely to be clinically significant.

Endometrial tissue and blood plasma concentration of ceftiofur and metabolites following intramuscular administration of ceftiofur crystalline free acid to mares

REASONS FOR PERFORMING STUDY Systemic administration of ceftiofur crystalline free acid (CCFA) may be a potential treatment for infectious endometritis caused by Streptococcus equi ssp. zooepidemicus (S.  zooepidemicus) and other susceptible bacterial organisms in the mare. OBJECTIVE To determine if i.m. administration of CCFA at the label dose will exceed the minimum inhibitory concentration (MIC) of S.  zooepidemicus in the endometrium following single administration and multiple administration protocols. STUDY DESIGN Experimental pharmacokinetic study. METHODS Three mares (Group 1) were administered a single i.m. dose of CCFA (6.6 mg/kg bwt) and blood and endometrial biopsies were collected at selected intervals for 144 h. Six additional mares (Groups 2 and 3) received CCFA at times 0, 4, 11 and 18 days, and were sampled at predetermined times for 25 or 49 days, respectively. Plasma and tissue samples were analysed by high-pressure liquid chromatography with tandem mass spectrometry for desfuroylceftiofur acetamide concentration, which is a direct measure of all ceftofur and ceftiofur metabolites in the sample. RESULTS A mean plasma desfuroylceftiofur acetamide concentration of 0.367 ± 0.0162 μg/ml (mean ± s.e.) was detected at 96 h following administration. Mean endometrial tissue concentration was 0.510 ± 0.0418 μg/g at 96 h and exceeded the MIC for S.  zooepidemicus (0.25 μg/ml) throughout the 144 h monitoring period for Group 1. Mares in Groups 2 and 3, given multiple doses of CCFA, maintained plasma concentrations above the MIC for S.  zooepidemicus for 25 days. Endometrial tissue levels remained above the MIC at most data collection points for 25 days. CONCLUSIONS Ceftiofur crystalline free acid reaches appropriate endometrial tissue values to exceed the MIC of S.  zooepidemicus, a common cause of bacterial endometritis. Therefore, CCFA should be effective in the treatment of equine bacterial endometritis caused by S.  zooepidemicus and other susceptible bacterial pathogens in the mare.

Drug exposure and clinical effect of transdermal mirtazapine in healthy young cats: a pilot study.

Objectives The objective of this study was to measure drug exposure and clinical effects after administration of transdermal mirtazapine (TMZ) in healthy cats. Methods Phase I: seven healthy research cats received (1) 3.75 mg and 7.5 mg TMZ once aurally with 48 h serum sampling (serum samples were obtained via the jugular catheter at 0, 0.5, 1, 2, 5, 9, 12, 24, 36 and 48 h); (2) 7.5 mg TMZ and placebo daily aurally for 6 days then 48 h serum sampling; (3) 1.88 mg mirtazapine orally once with serum sampling at 1, 4 and 8 h. Phase II: 20 client-owned cats were enrolled in a randomized, double-blind, placebo-controlled, three-way crossover clinical effect study. Treatments consisted of 6 days of aural 7.5 mg TMZ or placebo gel at home, and 1.88 mg mirtazapine orally once in the clinic. Owners documented appetite, rate of food ingestion, begging activity and vocalization daily at home. On day 6, food consumed, activity and vocalization were documented in hospital, and trough and peak serum mirtazapine levels were obtained. Serum mirtazapine and gel concentrations were measured using liquid chromatography/tandem mass spectrometry. Results Phase I: administration of TMZ achieved measureable serum mirtazapine concentrations. Area under the curve0–48 of multidose 7.5 mg TMZ was significantly higher than single-dose 1.88 mg oral mirtazapine (OMZ) (P = 0.02). Phase II: client-owned cats administered TMZ had a significant increase in appetite (P = 0.003), rate of food ingestion (P = 0.002), activity (P = 0.002), begging (P = 0.002) and vocalization (P = 0.002) at home. In hospital there was a significant increase in food ingested with both TMZ and OMZ compared with placebo (P <0.05). Gel concentrations ranged from 87%–119% of target dose. Conclusions and relevance TMZ 7.5 mg daily achieves measureable serum concentrations and produces significant appetite stimulation despite variance in compounded gel concentrations, but side effects denote a lower dose is indicated.

Pharmacokinetics and bioavailability of orbifloxacin oral suspension in New Zealand White rabbits (Oryctolagus cuniculus)

OBJECTIVE To evaluate the pharmacokinetics and bioavailability of 2 doses of orbifloxacin in rabbits. ANIMALS 6 healthy purpose-bred adult female New Zealand White rabbits (Oryctolagus cuniculus). PROCEDURES Each of 3 rabbits received orbifloxacin at either 10 or 20 mg/kg, PO. Then, after a 1-week washout period, they received the same dose IV. Blood samples were collected from each rabbit at 0, 0.25, 0.5, 1, 2, 4, 6, 12, and 24 hours after drug administration. Plasma orbifloxacin concentration was measured with liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were determined by noncompartmental analysis for data obtained following PO administration and noncompartmental and compartmental analyses for data obtained following IV administration. RESULTS Following oral administration, the mean ± SD peak plasma orbifloxacin concentration was 1.66 ± 0.51 μg/mL for rabbits administered the 10 mg/kg dose and 3.00 ± 0.97 μg/mL for rabbits administered the 20 mg/kg dose and was attained at 2 hours after drug administration. The mean ± SD half-life of orbifloxacin in plasma was 7.3 ± 1.1 hours for rabbits administered the 10 mg/kg dose and 8.6 ± 0.55 hours for rabbits administered the 20 mg/kg dose. Mean bioavailability was 52.5% for rabbits administered the 10 mg/kg dose and 46.5% for rabbits administered the 20 mg/kg dose. CONCLUSIONS AND CLINICAL RELEVANCE Results provided pharmacokinetic properties for 2 doses (10 mg/kg and 20 mg/kg) of orbifloxacin oral suspension in rabbits. Further studies are necessary to determine the protein-binding activity of orbifloxacin in rabbits before dosages for the treatment of common pathogens in this species are recommended.

The Effects of Mepivacaine Hydrochloride on Antimicrobial Activity and Mechanical Nociceptive Threshold During Amikacin Sulfate Regional Limb Perfusion in the Horse.

OBJECTIVE To determine the effect of intravenous regional limb perfusion (IVRLP) with a combination of mepivacaine hydrochloride and amikacin sulfate on synovial fluid amikacin sulfate concentration, antimicrobial activity, and mechanical nociceptive threshold (MNT). STUDY DESIGN Experimental study. ANIMALS Healthy adult horses (n=9). METHODS One IVRLP treatment was randomly administered by cephalic vein of each limb: amikacin alone (1 g amikacin in 60 mL saline) or amikacin with mepivacaine (1 g amikacin and 500 mg mepivacaine in 60 mL saline). Opposite treatments were repeated after a 24 hour wash-out period. Amikacin concentration and antimicrobial activity were determined for synovial fluid from middle carpal joints at tourniquet removal and 30 minutes following. Zone of inhibition was determined for Staphylococcus aureus and Escherichia coli. MNT was determined at 3 dorsal metacarpal locations prior to and after sedation, after Esmarch tourniquet application, and 30 minutes after IVRLP prior to and after tourniquet removal. RESULTS Two limbs from each treatment group were removed because of undetectable amikacin concentrations for a total of 14 data sets analyzed. Synovial fluid amikacin concentrations and zone of inhibition were not significantly different between treatments at any time point. MNT were significantly increased 30 minutes after IVRLP prior to and following tourniquet removal using amikacin and mepivacaine (median, range; 40.0 µg/mL, 38.7-40.0 and 40.0, 25.8-40.0, respectively) compared to amikacin alone (19.5 µg/mL, 18.7-25.6 and 15.3, 13.2-20.5, respectively). CONCLUSION Addition of mepivacaine to amikacin for IVRLP in the horse as a means of providing analgesia without decreasing antimicrobial activity.