Animal Clinical Investigation: Publications

PetMedicus Laboratories Receives MUMS Designation for
Ondansetron (VML-028) in Cisplatin-Induced Emesis

PetMedicus Laboratories, a client of ACI, was officially granted Minor Use and Minor Species (MUMS) status for their generic version of ondansetron (VML-028) for use in prevention of cisplatin-induced emesis on August 24, 2007 (http://www.fda.gov/cvm/MumsDesigList.htm). ACI played an important role in helping PetMedicus obtain this designation, representing only the second approved application for minor use in a major species of a Center for Veterinary Medicine (CVM) drug candidate. The designation was, in part, due to the completion of epidemiologic surveys by the ACI oncology network sites. These surveys provided essential population data that enabled the CVM to determine that the PetMedicus application fulfilled the requirements for minor use of a potential drug candidate.

Overview of MUMS

The US Food and Drug Administration (FDA) created the Minor Use and Minor Species Animal Health Act of 2004 (MUMS act) http://www.fda.gov/cvm/MUMSDrugDesg.htm. The MUMS act amended the Federal Food, Drug, and Cosmetic Act by, among other things, establishing section 573 to create new regulatory procedures that provide incentives intended to make more drugs legally available to veterinarians and animal owners for the treatment of minor animal species and uncommon diseases in major animal species. This Act parallels similar legislation passed under the Orphan Drug Act http://www.fda.gov/orphan/oda.htm for humans. The final rule implements section 573 of the MUMS act and describes the procedure for designating a new animal drug as a minor use or minor species drug. MUMS designation of a new animal drug grants drug sponsors seven years of exclusive marketing rights for these limited-demand drugs to encourage the commercial development of drugs for minor uses or in minor animal species.

Thanks to all of our ACI network sites for their participation and assistance in completing these invaluable surveys!

EVALUATION OF THE ORAL ANTIMITOTIC, ABT-751, ALONE AND IN COMBINATION AN ANTIANGIOGENIC PEPTIDE OF THROMBOSPONDIN-1, ABT-510, IN DOGS WITH LYMPHOMA

M Silver, T Rusk, C Khanna, Animal Clinical Investigation, Bethesda, Maryland 20817

E-mail address: msilver@animalci.com

Introduction: Previous studies defined the MTD of ABT-751 in dogs with lymphoma to be 350 mg/m2. Dose limiting toxicities (DLT) were gastrointestinal, including vomiting and diarrhea. Early evidence of clinical activity was observed at this dose. ABT-510, an oral antiangiogenic agent has resulted in efficacy in open-label treatment of dogs with a variety of cancers, including lymphoma.

Methods: Prospective studies of single-agent ABT-751 or in combination with ABT-510 were undertaken in dogs with lymphoma, most of whom had received previous chemotherapy. The dose of ABT-751 was 350mg/m2 PO QD for 7 days, then EOD. ABT-510 was given at 0.5mg/kg SQ BID starting 24 hours after the first dose of ABT-751. Primary endpoints included response rate, response duration, time to progression, and toxicity. Secondary endpoints included pharmacokinetics and changes in circulating endothelial cells.

Results: Treatment with single agent ABT-751 resulted DLT in 4/19 (21.0%) dogs. DLT was observed in 9/25 (36.0%) dogs treated with combination therapy. Cytopenias were observed in 3 dogs treated with combination therapy. No cytopenias were observed in dogs treated with ABT-751 alone. In the treatment-received population, which included dogs on study for at least 7 days, clinical responses were seen in 3/15 (20.0%) treated with ABT751 and 8/20 (40.0%) treated with ABT-751 and ABT-510. Response duration ranged from 8-111 days.

Conclusion: Though small study numbers were evaluated, response rate was higher for combination ABT-751 and ABT-510 versus ABT-751 alone. Toxicity profiles were similar for combination therapy and single-agent ABT-751. No previous toxicity has been observed in over 300 dogs treated with ABT-510; thus, toxicities seen with combination therapy are attributed to ABT-751.

A randomized placebo-controlled clinical trial of the anti-angiogenic thromobspondin-mimetic peptide ABT-526 plus Lomustine chemotherapy versus Lomustine chemotherapy alone in pet dogs with relapsed
non-Hodgkin’s lymphoma.

Chand Khanna, Elizabeth Cozzi, Richard Sharpee, David Vail, Joanne Graham, Barbara Kitchell, Tony Rusk

Background:

Thrombospondin-1 (TSP-1) is a natural antiangiogenic protein that enhances apoptosis of activated endothelial cells (EC). A modified nonapeptide from thrombospondin-1, ABT-526, has been found to be active in mouse cancer models and in dogs with naturally occurring cancers.

Methods:

To assess the safety and efficacy of ABT-526 when given in combination with Lomustine chemotherapy, 94 pet dogs with naturally occurring non-Hodgkin’s lymphoma (NHL), in their first relapse, were entered to a prospective randomized placebo controlled double-blinded clinical trial. Dogs were randomly assigned to receive ABT-526 plus Lomustine versus placebo plus Lomustine. Response rate, duration of response, time to progression, and incidence of toxicoses were compared between groups.

Results:

No significant ABT-526 specific toxicities were seen. Lomustine associated dose-limiting toxicities, including neutropenia, thrombocytopenia, gastroenteritis, and elevated alanine transaminase, were similar between treatment groups. No significant difference in the objective response rate was seen between treatment groups [ABT-526+Lomustine = 23/49 (47%) vs placebo+Lomustine = 23/37 (62%); P>0.25]. However, the median response duration was significantly greater in patients receiving ABT-526 plus Lomustine compared to placebo plus Lomustine (35 days vs 15 days; P=0.05). The time to progression for responding cases was also significantly greater in patients receiving ABT-526 plus Lomustine compared to placebo plus Lomustine (41 days vs 21 days; P=0.047).

Conclusions:

The significant activity of ABT-526 demonstrated in this preclinical trial appears to be associated with the maintenance of Lomustine induced treatment responses. Further studies of ABT-526, in this relevant naturally occurring model of NHL, are warranted and may be used to define biomarkers that predict responsiveness to antiangiogenic therapy and evaluate the activity of ABT-526 in combination with conventional and novel treatment agents.

A Randomized Placebo-Controlled Clinical Trial Of ABT-526 Antiangiogenic Therapy Plus Lomustine Chemotherapy Versus Lomustine Chemotherapy Alone In Dogs With Relapsed Lymphoma.
Anthony Rusk¹, Elizabeth Cozzi², Rick Sharpee², David Vail³, Joanne Graham⁴, Barbara Kitchell, Chand Khanna¹. Animal Cancer Institute LLC¹, Columbia, MD, Abbott Laboratories, Inc², N. Chicago, IL University of Wisconsin School of Veterinary Medicine³, Madison, WI Arboretum View Animal Hospital⁴, Downers Grove, IL, University of Illinois School of Veterinary Medicine⁵.

Thrombospondin-1 (TSP-1), a natural angiogenesis inhibitor, has been found to act broadly against a wide variety of proangiogenic growth factors. Its large molecular size and multifunctional nature has precluded its use as a therapeutic agent. Modified peptide segments of the antiangiogenic domain of TSP-1 containing D-amino acids have been shown to mimic the antiangiogenic action of TSP-1. Recently, a modified nonapeptide, ABT-526 (DI-TSPa) has been found to show significant antiangiogenic activity in vitro and in vivo. As a single agent ABT-526 treatment has resulted in objective tumor responses in histologically confirmed measurable tumors in pet dogs. Surprisingly, chemotherapy resistant lymphoma was found to be a responsive histology. Since the mechanism of action and targets for antiangiogenic agents and conventional cytotoxic agents are distinct, it is rationale to study combinations of these agents in the treatment of cancer.

Ninety-three dogs experiencing their first relapse of histologically confirmed lymphoma were randomized for study to a prospective, randomized, placebo-controlled, and double-blinded multicenter clinical trial to assess the safety and efficacy of ABT-526 when given in combination with Lomustine (CCNU) chemotherapy. Treatments assignments included ABT-526 + CCNU versus placebo + CCNU. Response rate, duration of response, time to progression, and prevalence of toxicoses, particularly number of episodes of dose-limiting neutropenia, thrombocytopenia, and both gastrointestinal and hepatic toxicoses were compared between groups.

No difference in response rate was seen between treatment groups. Median response duration and time to progression for responding cases was significantly greater in patients receiving ABT-526 plus CCNU compared to placebo plus CCNU (Median time to progression 41 vs 21 days; P=0.047). No differences in the incidence of dose-limiting episodes of neutropenia and gastrointestinal or hepatic toxicoses were seen between treatment groups.

The results of this study confirm significant biological activity for ABT-526 in dogs with relapsed lymphoma. The activity of ABT-526 demonstrated in this trial appears to be associated with the maintenance of CCNU induced treatment responses. The clinical value of ABT-526 in this treatment setting is of questionable significance due to the short CCNU induced response duration. Further studies to define the clinical utility of ABT-526 in combination with chemotherapy protocols that are able to induce more durable response durations and/or in the treatment of more responsive populations, i.e., dogs with newly diagnosed lymphoma, are warranted.

Antiangiogenic Thrombospondin-I peptides result in regression of naturally occurring cancers in pet dogs.
C. Khanna, T, Rusk, F. Haviv, and J. Henkin

Thrombospondin-1 (TSP-1) is a natural antiangiogenic protein that enhances apoptosis in activated endothelial cells (EC). D-amino acid substituted peptides within the TSP-1(Mal II) sequence inhibit EC function. A related antiangiogenic nonapeptide (ABT526) was found to slow tumor growth in syngeneic and xenograft mouse models. To examine the safety and efficacy of ABT526 antiangiogenic therapy a prospective pre-clinical trial in pet dogs with naturally occurring cancers was undertaken. Eligible cases had histologically confirmed measurable cancers, no cancer therapy within 21 days and no concurrent cancer therapy. The first 26 cases received 12.5 mg BID, SC; subsequent cases received 0.5 mg/kg BID, SC. Treatment was continued until significant progressive disease (SPD). Endpoints included adverse effects, significant disease stabilization and objective responses (PR >50%; CR 100% response) in measurable lesions. Seventy-four cases (carcinomas, lymphomas, sarcomas and others) were entered for study. Over 95% had failed conventional treatments prior to ABT526 monotherapy. Fifty-six dogs received at least 30 days of therapy before SPD (evaluable cases). Treatment was not associated with observable toxicity in any animal, and no disturbance of wound healing was seen in four cases requiring surgery. Unexpected disease stabilization and objective responses were seen in 11/56 and 8/56 evaluable cases, respectively. Relapse after durable objective responses were seen in 8/8 cases.

Objective responders with ABT526 antiangiogenic therapy

Histology	Clinical Stage	Response
Nasal Carcinoma	T3bN0M0	CR
Cutaneous Lymphoma	Generalized	CR
Nasal Carcinoma	T3bN1M0	PR
NH Lymphoma	IIIb	PR
Soft Tissue Sarcoma	T2bN2N3bM1(lung)	PR
Cutaneous Lymphoma	Generalized	PR
GI adenocarcinoma	TsxN2N1M0	PR
NH Lymphoma	IIIa	PR

Results suggest that ABT526 therapy is well-tolerated, effective, and associated with regression of measurable lesions in naturally occurring cancers. A randomized trial of ABT-526 plus chemotherapy versus chemotherapy alone in canine NH lymphoma is underway.

Objective respon der s w ith ABT526 antiangiogenic therapy include (Histology- Clinical Stage-Response): Nasal Carcinoma-T3bN0M0-CR; Cutaneous Lymphoma- Generalized-CR; Nasal Carcinoma-T3bN1M0-PR; NH Lymphoma-IIIb-PR; Soft Tissue Sarcoma-T2bN2N3bM1(lung)-PR; Cutaneous Lympho ma-Generalized-PR; GI-adenocarcinoma-TsxN2N1M0-PR; NH Lymphoma-IIIa-PR.

Tumor inhibition by antiangiogenic TSP-I mimetic peptides
Poster presentation - American Association of Cancer Research 2002
Jack Henkin, Fortuna Haviv, Yi-Chun Wang, David Frost, Abdullah Kherzai, Frank K. Reiber, Olga V. Volpert, Susan E. Crawford, Noel P. Bouck, Steven C. Campbell, Chand Khanna

A new option for the treatment of canine lymphoma
Non peer reviewed summary, 2/2002
Chand Khanna DVM, PhD, Dipl ACVIM (Oncology)
Animal Cancer Institute LLC

Lymphoma is the most common malignant cancer treated in veterinary oncology. In dogs, the most common presentation for lymphoma is multiple non-painful enlargements of peripheral lymph nodes. It is most common that dogs with lymphoma present without any clinical signs of illness in spite of these enlarged lymph nodes. In most cases lymphoma is thought of as a disseminated disease, where the malignant lymphocytes exist in many tissues through the body. The diagnosis of lymphoma is made by microscopic examination of lymph nodes. Unlike many other cancers, a diagnosis of lymphoma can be made using a fine needle aspirate alone. Microscopic features that distinguish malignant lymphocytes from normal lymphocytes include their larger size, multiple and variably sized nucleoli, and more frequent mitotic figures.

Treatment of Canine Lymphoma

Over time lymphoma will progress to involve both lymphoid and non-lymphoid organs. As the disease spreads and involves vital organs a progressive decline in quality of life is noted. The progression of this cancer often occurs quickly in dogs, but is quite variable. Lymphoma is one of the most chemo-responsive cancers in dogs. Dogs treated with chemotherapy can have a very high quality of life for many months to years. Dogs that feel well when therapy is initiated have the best chance of successful treatments. Dogs that respond well following the first one to two treatments often enjoy longer remission durations.

Options for the treatment of lymphoma include multi-agent chemotherapy, single agent chemotherapy, and prednisone alone. In addition, a new treatment is now available.

Multi-agent chemotherapy:
The use of multi-agent chemotherapy is expected to result in an initial complete regression (remission) of lymphoma in over 80% of dogs. On average this first remission will last for approximately one year. The return of the enlarged lymph nodes often marks the end of the remission. The following is an example of a multiple agent chemotherapeutic protocol used to treat canine lymphoma.

Canine Lymphoma Multi-Agent Chemotherapy Protocol (Khanna et al 1996 JAVMA).

Single agent chemotherapy for lymphoma
Single agent chemotherapy protocols have been developed for lymphoma. Single agent therapies include doxorubicin, mitoxantrone and lomustine. Single agent doxorubicin chemotherapy is expected to provide a first remission rate that is similar to multiagent protocols and a first remission duration of approximately 7 months. Doxorubicin is administered as a single agent every 21 days for a total of 3 - 5 treatments.

Prednisone therapy for lymphoma
Prednisone alone may result in a regression of measurable lymph nodes in approximately 50% of cases. The duration of remission associated with prednisone has been reported to be 75 to 90 days. Prednisone doses for dogs with lymphoma are 40 mg/m2 daily.

The advantage of multi-agent chemotherapy for lymphoma is longer remission durations that result from the combination of drugs with different mechanisms of action. Although combination protocols have been designed to maximize quality of life, they are associated with a greater risk for toxicity tha single agent protocols. Clients who are concerned by the intensity of multi-agent protocols may be more comfortable with single agent therapies. Single agent therapies also have the advantage of being less expensive. Prednisone alone is often considered the best option for owners who are unable to consider chemotherapy due to financial restraints.

Recently a novel chemotherapy has become commercially available and is approved for the treatment of both pancreatic and lung cancer in people. This agent is in a class of chemotherapies referred to as a nucleoside analogue. Nucleoside analogue disrupt the synthesis of RNA in rapidly dividing cells. The activity of this new agent in canine cancer is largely anecdotal and unknown. We have treated a number of cancers in pet dogs using this agent, including lymphoma, soft tissue sarcoma, osteosarcoma, and hepatic carcinoma. The responses observed to date have been encouraging and suggest the need for further evaluation of this drug in pet dogs. Interestingly this agent has been associated with a very favorable toxicity profile in all cases treated to date. To determine if this new nucleoside analogue is effective in lymphoma a clinical trial has been designed and will open in April 2002 at a number of treatment sites in the United States. This trial is open for pet dogs with lymphoma that have not received prior chemotherapy. The trial provides all costs associated with diagnosis, treatment and follow up for dogs. This trial may be an ideal option for owners that are concerned about the side effects of conventional chemotherapy or find the costs associated with chemotherapy burdensome. For more information about this clinical trial please contact Kate Cadorette.

Targeting the blood supply of cancer. Discussion of novel and non-toxic treatment available for pet dogs with lymphoma
Chand Khanna DVM, PhD, Diplomat ACVIM (Oncology)
Animal Cancer Institute, LLC

New, effective, and non-toxic treatments are now under investigation in both pet animals and people with cancer. These new treatments have become available through a dramatic increase in our understanding of the basic biology of cancer and the requirements for cancer growth and spread. An essential requirement for cancer growth and spread appears to be the development of new blood vessels (angiogenesis). If a cancer requires new blood vessels in order to progress, then it may be possible to prevent a cancer from growing or spreading by preventing it from creating new blood vessels. This possibility is now the basis of a novel approach to the treatment of cancer that is based on either preventing a cancer from creating new blood vessels (antiangiogenic agents) or specifically destroying the blood vessels in a cancer.

Angiogenesis is a complex and intricately controlled process. This process is based on a balance between positive factors that promote new blood vessel formation and negative factors that inhibit new blood vessel formation. The goal of new anti-angiogenic therapies is to shift this balance, which in cancer has "switched" towards new blood vessel formation, in the other direction, i.e. towards the inhibition of blood vessel formation. Many factors have been found to influence this blood vessel formation balance. Several are currently under investigation as potential anticancer agents.

Since most adult tissues do not require new blood vessel formation, these new anticancer agents hold the promise of effectiveness and the absence of significant toxicity. Tissues in which new blood vessel formation is needed in the adult include healing wounds, the outer surface of the eye (cornea), the uterine lining during the estrus cycle, and joints that are responding to arthritis. It appears that the normal regulation of blood vessel formation in these tissues and conditions is different from the new blood vessel formation seen in cancer. This may explain why many antiangiogenic agents have not been associated with problems such as wound healing, arthritis, or keratitis (eye irritation).

Recent studies have demonstrated that in addition to differences in the regulation of new blood vessel formation in cancer compared with normal tissues, the actual blood vessels that are "created" in cancers are different from those created in normal tissues. These differences have allowed a number of new agents to be developed that specifically damage tumor-associated blood vessels and not normal vessels. The goal of these agents is to attack cancers by damaging their blood supply. Many agents that inhibit blood vessel formation (antiangiogenic agents) also appear to hasten the death of tumor-associated blood vessels.

Thrombospondin-I is a large protein that has a number of biological roles including the inhibition of new blood vessel formation and the enhancement of existing blood vessels death. These biological roles have suggested the potential value of Thrombospondin-I as a potential anti-cancer agent.

The diverse roles of Thrombospondin-I during angiogenesis

Recent studies have focused on small fragments of Thrombospondin-I that are thought to be specifically associated with angiogenesis. These fragments have been shown to be potent inhibitors of new blood vessel formation and appear to promote the death (apoptosis) of blood vessel cells.

Over the last 2 years the Animal Cancer Institute has been evaluating the safety and activity of these small fragments of Thrombospondin-I in pet dogs with malignant cancers. At the onset of this ongoing trial we were hopeful that Thrombospondin-I peptides would control the growth of measurable cancers in pet dogs and result in disease stabilization. We have been surprised to find that not only did control of disease occur in some dogs but that a small proportion of pet dogs had objective regressions of their cancers. Regressions of cancers were seen in both primary tumors and in cancers that had spread to other parts of the body (metastases). Results from a series of dogs treated with a Thrombospondin-I fragment will be presented at the annual meeting of the American Association for Cancer Research in San Francisco in April and are expected to be presented at the annual meeting of the American Society of Clinical Oncology in Orlando in May. For more information on this ongoing clinical trial please contact Kate Cadorette at kcadorette@animalcancerinstitute.com.

Barney a 13 year old black lab who was diagnosed with a nasal sarcoma. The tumor had been treated with both radiation therapy and chemotherapy but was recurrent. Prior to receiving Thrombospondin-I Barney was experiencing life threatening nasal bleeding on a daily basis. Treatment with Thrombospondin-I resulted in resolution of nasal bleeding and a complete regression of a portion of the tumor that had invaded the hard palate. Barney began breathing comfortably through his nose and continued to do well on Thrombospondin-I for approximately 8 months.

One of the interesting results from the clinical trials using Thrombospondin-I peptides has been their activity against canine lymphoma. This preliminary evidence of effectiveness against lymphoma has supported the initiation of a randomized trial of Thrombospondin-I peptides in combination with Lomustine chemotherapy for dogs with lymphoma. This trial provides conventional chemotherapy to all dogs and then the addition of the active Thrombospondin-I peptides or placebo in a randomized manner. In this way all dogs receive a standard and appropriate treatment for lymphoma (i.e. Lomustine chemotherapy) and then may additionally receive the antiangiogenic therapy. The clinical trial supports the majority of the cost for treatment, laboratory testing, and follow-up. Dogs eligible for this trial must have a relapse of B cell lymphoma that was initially treated with conventional chemotherapy. This trial is currently available through a number of veterinary referral hospitals and universities in the United States. For more information please contact Kate Cadorette at kcadorette@animalcancerinstitute.com.

Agents that inhibit new blood vessel formation or specifically target tumor-associated blood vessels represent a novel, potentially effective, and non-toxic treatment for cancer. It is likely that these agents will provide the next major breakthrough in the management of pet animals and people with cancer. Antiangiogenic therapy will likely become part of the conventional treatment of cancer and will be used in combination with surgery, radiation therapy and chemotherapy. These agents are currently in clinical trials and may become available to both pet animals and people in the near future.

ONGOING EVALUATION OF SINGLE AGENT THALIDOMIDE IN DOGS WITH MEASURABLE CANCER.

Jankowski M¹, Fulton L², Sheafor S³, Prescott D², Khanna C.^1,4 - Friendship Hospital for Animals1, Washington D.C., VCA-Veterinary Referral Associates², Gaithersburg MD, SouthPaws Veterinary Specialists and Emergency Center³, Springfield VA, Pediatric Oncology Branch⁴, NCI-NIH, Bethesda MD.

INTRODUCTION
Angiogenesis is essential for cancer progression and metastasis. Inhibition of angiogenesis may be an effective treatment for cancers. The anticancer activity of thalidomide has been demonstrated in early human clinical trials. The objectives of this study are: 1) to determine the safety and efficacy of oral thalidomide against measurable canine cancers, 2) to define an optimal biological dose for thalidomide in dogs, and 3) to define tumor types sensitive to antiangiogenic therapy with thalidomide.

METHODS
This is a single agent, phase I/II clinical study. Entry requirements include measurable and histologically confirmed disease, body weight > 10 kg, no chemotherapy within 10 days of treatment, and informed owner consent. Three escalating dose cohorts (30 cases/cohort) have been defined: 3.3-6.5, 6.6-13, and 13.3-26 mg/kg QD. Physical examinations, CBC, serum biochemistry, and tumor response will be evaluated at day 0 and every 30 days thereafter. Urine and plasma samples collected before and during thalidomide therapy will be assayed for markers of antiangiogenic activity including, basic fibroblast growth factor, vascular endothelial growth factor and interleukin-8.

RESULTS
Six cases to date have been evaluated. All dogs had progressive disease prior to study entry. No side effects to thalidomide have been noted in the first dose cohort.

Histology	TNM Stage	Measurable Lesion	Prior Treatment	Treatment Length	Outcome
Malignant pericardial mesothelioma	TsxM1pulmonary (possible CNS)	Pulmonary	Surgery, Chemotherapy, Immunotherapy	116 days	SD x 90 days then PD
Nasal carcinoma	T3N2mandibular, prescapular M0	Lymph nodes, Orbital proptosis	Radiation therapy, Chemotherapy	80 days	SD x 77 days then PD
Rib osteosarcoma	T2M1 pulmonary	Pulmonary, local recurrence	Surgery, Chemotherapy	33 days	PD
Carcinomatosis abdomen		Liver	None	27 days	PD
Hepatocellular, carcinoma		Liver	Surgery	36 days	PD
Osteosarcoma	T2M1 pulmonary	Pulmonary	Surgery, Chemotherapy	9 days	PD

DISCUSSION
Thalidomide appears be well tolerated in the current dose cohort. No objective tumor responses have been documented at this time. Additional entry of cases with smaller tumor burden may allow antiangiogenic activity to be exerted before disease progression. Surrogate markers of systemic antiangiogenic activity, measured in plasma and urine, may be helpful in defining optimal biologic dose.