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Keywords:

  • Chemotherapy;
  • Epothilones;
  • Microtubule-stabilizing agents

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background

The epothilones are microtubule-stabilizing agents with promising antitumor effect in refractory and metastatic tumors in humans. The toxicity profile is considered more favorable than in taxanes. The safety of epothilone B (patupilone) has not been evaluated in tumor-bearing dogs.

Objectives

To evaluate the inhibition of proliferation in canine tumor cells after patupilone treatment. To assess toxicity profile and maximally tolerated dose of patupilone in dogs with refractory tumors.

Animals

Twenty client-owned dogs with various malignancies.

Methods

Prospective clinical study. The inhibition of proliferation was assessed with a proliferation assay in vitro in canine hemangiosarcoma and lymphoma cell lines. Dogs received patupilone IV once a week for 2 treatments (= 1 treatment cycle). Dose was escalated with 3 dogs per cohort and 20% increments. Adverse effects were graded accor-ding to the VCOG-CTCAE v1.0.

Results

Both canine cell lines were sensitive to patupilone with approximately 50% decrease in proliferative activity at 0.2–1 nM. In vivo, dose-limiting adverse effects occurred at 3.3 mg/m2; main adverse effects were diarrhea, anorexia, vomiting, and nausea. Neither neutropenia nor peripheral neuropathy was observed. Maximally tolerated dose for 2 patupilone administrations once weekly IV is 2.76 mg/m2. Three per 11 dogs receiving more than 1 treatment cycle showed partial remission in the short period of observation.

Conclusions and Clinical Importance

Canine tumor cells show inhibition of proliferation to patupilone in vitro. Clinically, a dose of 2.76 mg/m2 IV is well tolerated in dogs with spontaneously occurring tumors.

Abbreviations
AE

adverse effects

CR

complete response

MSA

microtubule-stabilizing agents

MTD

maximally tolerated dose

PD

progressive disease

PR

partial response

SD

stable disease

Epothilones are potent nontaxoid microtubule-stabilizing agents (MSA) of bacterial origin sharing the binding site on β-tubulin with the taxanes, which belong to the most potent antineoplastic agents.[1, 2] MSA interfere with microtubule function in the proliferating cell by preventing the shortening of microtubules, resulting in a permanent or transient cell cycle arrest at the G2/M-phase, which leads to apoptosis.[1, 2] Ixabepilone1 is the 1st compound of the epothilones to be approved as monotherapy or in combination with capecitabine for the treatment of women with metastatic breast cancer, and demonstrates activity in tumors that show resistance toward anthracycline and taxane standard therapy.[2, 3] Compared with the taxanes, epothilones are described to be more potent and possess several advantages such as increased solubility, low susceptibility to drug efflux transporters (eg, p-glycoprotein-mediated chemoresistance), and a more tolerable toxicity profile in human patients, which favor their development and make them promising agents for further use in dogs as well.[2, 4] Patupilone (EPO906) is a natural, water-soluble epothilone B extracted from mycobacterium sorangium cellulosum and is the most active variant of the natural epothilones. The efficacy of patupilone is based on not only targeting tumor cells but also the vasculature and angiogenesis of solid tumors.[5-7] Patupilone was tested as a phase III monotherapy agent against ovarian cancer in human patients and other epothilones are currently undergoing a wide spectrum of single and combined treatment modality in phase II and phase III studies (eg, for recurrent glioblastoma multiforme, breast cancer brain metastases, prostate, cervical, renal cell, gastric and lung tumors, as well as non-Hodgkin's lymphoma).[8-12]

To introduce this promising antineoplastic substance group into veterinary medicine, the objectives of this study were (1) to evaluate whether patupilone shows in vitro efficacy against canine tumor cell lines and (2) to determine the adverse effect (AE) profile and the maximally tolerated dose (MTD) for IV administration in dogs with various spontaneously occurring, treatment-refractory tumors.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In Vitro Study

Canine Tumor Cell Lines

A canine malignant T-cell lymphoma cell line (CL-1) and a canine hemangiosarcoma cell line were used.[13, 14] CL-1 cells were cultured in RPMI 1640 with 1% (v/v) stable glutamine,2 10% (v/v) fetal bovine serum,3 and 1% (v/v) penicillin-streptomycin.4 Hemangiosarcoma cells were cultured in D-MEM/F-12 containing 1% (v/v) L-glutamine,5 10% (v/v) fetal bovine serum,6 and 1% (v/v) antibiotic-antimycotic7 solution. All cells were incubated at 37°C in a humified incubator with 5% v/v CO2.

Study Drug

Patupilone (epothilone B) was provided by Novartis Pharma, Basel, Switzerland. To obtain the stock solution, patupilone was dissolved in DMSO to a concentration of 1 mM and further stored at 2–8°C. Immediately before addition to cell lines, stock solution was diluted in culture medium to achieve the appropriate concentrations. Final DMSO concentrations used in the assays were in the nanomolar range (≤0.0001%) and were considered negligible.

Cell Proliferation Assays

The proliferative activity of tumor cells was assessed with the colorimetric alamarBlue® assay8 according to the protocol of the manufacturer. AlamarBlue® assesses metabolic activity of living cells by measurement of spectral absorption. Proliferation assays were performed in 96-well plates with 1 × 104 and 0.75 × 104 cells/well for CL-1 and hemangiosarcoma cell lines, respectively. Before the experiment, optimization assays (cell number) were performed to optimize the absorbance reading for spontaneous proliferation (ie, proliferation at 0 nM). Each well contained 100 μL of cell suspension in the concentration mentioned above, wells used as blank controls contained 100 μL of media only. After a 24-hour incubation period, the cells were treated with 10 μL of increasing concentrations of patupilone, control cells (0 nM) and blank wells (media only) were treated with 10 μL of plain medium. Absorbance was measured in triplicates before and 24, 48, and 72 hours after patupilone treatment at 570 and 630 nm using the spectrophotometer SpectraMax® Plus384 Absorbance Microplate Reader9 and SoftMax® Pro 4.8 Software10 after a 6-hour incubation period of alamarBlue® dye. Background absorbance of cell media was subtracted. All assays were repeated as independent experiments at least thrice. The approximately 50% decrease in proliferative activity was derived from graphical plotting of data.

In Vivo Study

Patient Eligibility Criteria

Dogs presented to the Vetsuisse Faculty, University of Zurich, Switzerland with histologically or cytologically confirmed neoplastic disease were prospectively enrolled in this study if they were refractory to standard of care treatment, if the owner declined other treatment, or if no established standard of care existed. To meet inclusion criteria, dogs required a modified Karnofsky's score of ≤1, adequate hematologic, renal, and hepatic function and absence of any serious systemic disorder incompatible with the study at the discretion of the investigators.[15] Prior chemotherapy treatment had to be terminated at least 2 weeks before start of the study. Written owner consent was obtained and the study was approved by the Animal Ethics Council of the Canton of Zurich, Switzerland.

Study Design and Drug Administration

This trial was a phase I dose-escalating, open-label assessment of adverse effects (AE) of patupilone in client-owned dogs with spontaneously occurring tumors. Safety was assessed on enrollment of at least 3 dogs into dose-escalating cohorts using a traditional 3+3 design.[16, 17] Escalation was performed according to Vail[17] where the dose was escalated if none of 3 dogs of 1 dose cohort developed dose-limiting AE as defined according to the VCOG-CTCAE v1.0.[18] If one of the 3 dogs of a cohort experienced a dose-limiting AE or if the 2nd treatment had to be delayed ≥5 days because of grade 2 toxicosis, then at least 3 more dogs were included at the same dose level. Dose escalation was stopped, if two or more dogs of a cohort of three, or an extended cohort of 6 patients, respectively, developed dose-limiting AE or a treatment delay. This dose level was the maximally administered dose. The MTD was defined as the highest dose level in which no more than one of 6 dogs develops a dose-limiting AE.[17] Escalation steps occurred in increments of 20% of the previous dose. Starting dose was 1.6 mg/m2, based on 50% of the MTD of experimental Beagle dog data.

Treatment schedule consisted of 2 IV administrations of the drug once weekly (= 1 treatment cycle). In turn, dogs were evaluated every week for adverse effects and tumor response. If no progression was noted, treatment with patupilone was continued at the same dose level.

Patupilone (epothilone B) for in vivo use was provided by Novartis Pharma, Basel, Switzerland as vials concentrate liquid of 10 mg/4 mL. To prepare the epothilone B for administration, the concentrate was diluted with normal saline, to obtain a concentration of 1 mg/mL. The calculated dose was given via IV infusion in parallel to 100 mL 0.9% saline over a 10- to 20-minute interval.

Supportive care (eg, antiemetics, antidiarrheals) was administered after occurrence of AE and was provided prophylactically after the next administration of drug. Medication for disease unrelated comorbidity or further supportive care including steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) that had been administered at study entry and under which the dogs had shown progressive disease before study entry were continued. Antibiotics were prescribed in case of ulceration of tumor, analgesics for tumor-associated pain were allowed, and dogs with mast cell tumors at risk of histamine release received H1- and H2-blocking agents.

Assessment of Adverse Effects

Dogs were evaluated for signs of intoxication weekly before each treatment and 1 week after the last chemotherapy. Evaluation of severity of toxicoses to determine MTD was carried out after the 1st and 2nd patupilone dose only. AE were monitored by performing a physical examination, complete blood count (CBC), and serum biochemistry at each scheduled appointment and by grading the dogs according to the Modified Karnofsky's Performance Criteria.[15] Owners were asked to keep a log at home. Blood results and signs of AE were graded according to the VCOG-CTCAE v1.0.[18] Unacceptable AE leading to discontinuation of the trial was defined as ≥grade 3 toxicosis, except hematological AE, where ≥grade 4 was considered unacceptable. Because this trial included heavily pretreated patients with an advanced stage of disease, a baseline status using the same criteria was recorded for each dog before start of the trial.

Tumor Response Assessment

Because of the dose-escalating nature of this trial, response to treatment was only a secondary goal. Tumor size (locoregional and systemic) was assessed at study entry, before each drug administration and 1 week after the last treatment by means of caliper measurements and additional diagnostic imaging according to the tumor. In nonmetastatic tumors, clinical staging (thoracic radiographs, abdominal ultrasound) was performed at study entry and comparable re-evaluations took place after 2 and 4 patupilone administrations to exclude disease progression by means of systemic extension. Response was assessed as follows: in dogs with multicentric lymphoma, criteria according to VCOG consensus document for peripheral nodal lymphoma in dogs were used.[19] Lymph nodes were measured using calipers and the mean sum of the longest diameter of a maximum of 5 target lesions was calculated. In dogs with other solid tumors, response was measured according to the revised RECIST guideline v1.1.[20] The sum of diameters of all target lesions was calculated and reported as the baseline sum diameter. According to both guidelines, responses were considered to be complete if all target lesions had disappeared (or lymph nodes were of normal size, respectively), partial (PR) if a 30% decrease in the sum of target lesion diameters occurred, or progressive (PD) if new lesions occurred or if there was an increase of at least 20% in the sum of target lesion diameters, taking as reference the smallest sum on study.[21] Dogs that neither showed sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD were considered to have stable disease (SD).[19, 20]

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In Vitro Study

Cell Proliferation Assays

In the 2 canine cell lines tested, 50% decrease in proliferative activity was found to be in the range of 1 nM in canine hemangiosarcoma and 0.2 nM in malignant lymphoma cells (Fig 1). Compared with drug-sensitive human cell lines, the tested cell lines showed sensitivity to patupilone.[22, 23]

image

Figure 1. Proliferation activity after patupilone treatment was assessed over 72 hours using the alamarBlue® assay. Canine hemangiosarcoma (A) and malignant lymphoma cells (B) showed a 50% decrease in proliferative activity at 1 and 0.2 nM, respectively. Both graphs show a single representative experiment with inclusion of standard deviation. a.u.: artificial unit.

Download figure to PowerPoint

In Vivo Study

Patient Characteristics

A total of 20 client-owned dogs were included in the study between July 2009 and October 2011. Patient population consisted of dogs of various breeds: mixed breed (n = 6), Boxer (n = 3), Golden Retriever (n = 2), American Pitbull, Jack Russell Terrier, Australian, Belgian, and German Shepherd, Doberman Pinscher, Greater Swiss Mountain Dog, Rottweiler, and Standard Poodle (1 each). Median age upon study entry was 8.75 years (range 6–14 years) and median weight 28.4 kg (range 5.7–51.4 kg). Four dogs were intact females, 2 intact males, 10 were spayed females and 4 castrated males. Different types of tumors were included; the most common primary tumors were malignant mammary carcinomas (n = 6), and malignant multicentric lymphoma (n = 6); two of the mammary tumors were diagnosed as anaplastic mammary adenocarcinoma. Other tumors included mast cell tumors (n = 3), prostate carcinoma (n = 2), anal gland adenocarcinoma, oral malignant melanoma, and cardiac hemangiosarcoma (1 each). The majority of dogs had received prior treatment with either chemotherapy (n = 10), surgery (n = 4), surgery and chemotherapy (n = 1), or radiation therapy (n = 1). All but 1 dog had measurable disease at the onset of patupilone treatment; 13/20 (65%) showed macroscopic local or locoregional disease (1 distant metastasis); 6/20 (30%) had multicentric malignant lymphoma; and one dog (5%) showed microscopic disease after surgery of a large invasive mammary carcinoma. All 6 dogs with malignant lymphoma showed multicentric involvement stages IV–V, substage a-b disease, and clinical resistance toward several previous chemotherapy protocols. Two dogs showed T-cell, 2 B-cell, 1 null-cell lymphoma, and in 1 dog, immunophenotyping was not performed.

Drug Administration

Patients were divided into 6 different dose groups from 1.6 to 3.3 mg/m2 with increments of 20%, except in the dose group 6 (see Table 1). At the starting dose of 1.6 mg/m2, only 1 dog was included; because of rapid disease progression and no drug-related AE, the 2nd dog was treated with the next higher dose. The other groups included at least 3 dogs per dose level, with additional 3 (confirmatory) patients at the MTD level. In total, 62 patupilone doses were administered, with a median of three per dog (range one to seven). Eighteen dogs received 1 cycle of patupilone (as planned) and eleven of these continued treatment with the study drug. Two dogs received 1 patupilone treatment only, because of grade 2 AE and owner's decline for further treatment in 1 dog, and grade 3 AE and exclusion of the study in another dog. Seven dogs received concurrent NSAIDs and eight received steroids.

Table 1. Gastrointestinal adverse effects after patupilone treatment as classified by VCOG-CTCAE v1.0.[18]
Dose GroupDose (mg/m2)Dogs (n)DiarrheaAnorexiaNauseaVomiting
Grade 1 + 2 (n)Grade 3 (n)Grade 1 + 2 (n)Grade 3 (n)Grade 1 + 2 (n)Grade 3 (n)Grade 1 + 2 (n)Grade 3 (n)
  1. a

    Dose was reduced by 10% to approach the optimal dose more precisely.

  2. b

    of which 2 were grade 2 leading to a relevant treatment delay of ≥5 days.

  3. c

    of which 1 was grade 2 leading to a relevant treatment delay of ≥5 days.

11.6110101010
21.9320202020
32.3320202000
42.766302c03050
53.3311022020
63a44b02c13040
Total 2014193130140
Assessment of Adverse Effects

Metabolic AE were evaluable after 60 of the 62 drug administrations; data of all other AE were assessable after every patupilone administration.

Metabolic: Hematological and serum biochemical AE were mild, as summarized in Table 2. In 9/13 dogs with anemia (17 administrations), grade 1 toxicosis was already present at study entry with no change during the study. Grade 1–2 thrombocytopenia occurred in 3 dogs with advanced malignant lymphoma; one of them had grade 1 impairment already at baseline and progressed to grade 2 after chemotherapy. No neutropenia was seen in any dog. All dogs with increase in alanine aminotransferase (ALAT) had received steroids for a prolonged period of time before study entry. Three dogs showed grade 3 increase in ALAT already at baseline and 3 dogs showed improvement or normalization of ALAT values at the end of chemotherapy.

Table 2. Metabolic adverse effects according to VCOG-CTCAE v1.0 after all 60 evaluable drug administrations.[18]
 Highest Toxicity GradeToxicities [Occurrence of AE; % of All Administrations]
  1. AE: adverse effects.

  2. a

    Grade 3 toxicosis existed already at study entry.

Anemia123 (38%)
Thrombocytopenia23 (5%)
Neutropenia00 (0%)
Hypoalbuminemia12 (3%)
Increase in alanine aminotransferase3a10 (17%)

Gastrointestinal: Main AE in this study were diarrhea, anorexia, nausea, and vomiting. Dose-limiting AE was determined to be anorexia and diarrhea. In affected dogs, diarrhea started with a median of 4 days after chemotherapy. Upon occurrence of gastrointestinal AE after the 1st drug administration, supportive care was administered in 14/20 (70%) dogs. Not all AE were considered to be attributed to patupilone administration: in some dogs, toxicoses were considered to be attributed to disease progression or were considered to be because of treatment-unrelated comorbidities. However, all AE regardless of cause are listed in Table 1.

Other: Grade 1–2 fatigue was noted after 18/62 (29%) drug administrations. In 2 dogs, grade 1 fatigue was already present at study entry. A modified Karnofsky's performance status score of 1–2 was seen after 15/62 (24%) treatments. In 2 dogs, however, a performance status of 1 was already recorded at baseline. No other AE was noted.

Maximally Tolerated Dose

Patupilone was well tolerated up to a dose of 2.76 mg/m2. At the next higher dose level of 3.3 mg/m2, 2 dogs showed dose-limiting grade 3 anorexia or anorexia and diarrhea, respectively. Dose was reduced by 10% to approach the optimal dose more precisely. At 3 mg/m2, only 1 dog showed dose-limiting AE, but the second application of chemotherapy had to be delayed for 1 week in two of the other dogs treated at this dose level because of grade 2 diarrhea or grade 2 diarrhea and anorexia, respectively. Therefore, MTD for 1 cycle of patupilone was determined to be 2.76 mg/m2.

Tumor Response Assessment

Response was evaluated in 18/20 dogs. At the end of all patupilone treatments (range one to seven), 3 dogs (16.7%) showed PR, 6 dogs (33.3%) SD, and 9 PD (50%). The 3/11 dogs receiving more than 1 treatment cycle showed partial remission in the short period of observation and included a dog with prostate carcinoma and 2 dogs with anaplastic mammary carcinoma and sublumbar lymph node metastases. In the latter, surgical excision of the primary tumor was performed 2 weeks before study entry. Both dogs showed recurrence at the site of surgery and experienced PR after administration of more than 2 patupilone administrations.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

According to this phase I trial, the administration of patupilone up to an IV dose of 2.76 mg/m2 was found to be safe in dogs with spontaneously occurring tumors and no hypersensitivity reactions were observed. This is in contrast to taxoid MSA such as paclitaxel and docetaxel where the use in dogs is limited because of the requirement of organic solvents that cause unwanted adverse effects even in dogs that are heavily premedicated.[24-26]

In the in vitro part of this trial, canine tumor cells showed a strong decrease in proliferative activity with low doses of patupilone (1 and 0.2 nM in canine hemangiosarcoma and lymphoma cells, respectively), when compared with known patupilone-sensitive and -resistant human cell lines.[23] In humans, a patupilone dose of 10 nM is considered to be an in vitro approximation of plasma levels at a MTD of 2.5 mg/m2.[6, 27] In an in vivo model with human colon tumor xenografts, patupilone administration resulted in high concentration in tumor tissue at 168 hours after chemotherapy, while other tissues showed decreased concentrations.[22] The presence of continuous drug exposure might contribute to tumor cell cytotoxicity and add to the prior-described antiangiogenic efficacy of the drug.[6, 23, 28] The MTD for 2 patupilone IV administrations given once weekly is 2.76 mg/m2, comparable to the dose in human patients for weekly administration.[29] If patupilone is given at longer intervals, a higher dose is tolerated in human, as the severity of gastrointestinal signs can be controlled by proactive, aggressive diarrhea-management.[30] However, the main goal of this trial was to find MTD after 1 treatment cycle of patupilone (2 administrations). One reason for the chosen schedule of 2 administrations aimed at being able to exploit not only the cytotoxic effect of the drug but also make use of the antiangiogenic and antivascular effects of epothilone B. Agents with antiangiogenic effects exert their efficacy after repeated administration of low doses with short interval in between, for example, daily or weekly treatment.[31] A 2nd reason for this schedule was the intention to use the drug as a radiosensitizer in combined treatment with ionizing radiation. Epothilones are considered to be optimal radiosensitizing drugs as they induce cell cycle block in the most radiosensitive G2/M-phase already at low and noncytotoxic doses. This led to additive or even supra-additive effect on tumor growth in mice with human cancer xenografts.[23, 32, 33]

Hematological AE were generally mild and no neutropenia was seen. One treatment cycle of patupilone is therefore not associated with clinically significant myelosuppression in dogs. The clinically relevant toxicosis in this study (≥grade 2) was gastrointestinal, seen in 50% of all patients with main AE anorexia, diarrhea, or both. However, in 6 dogs, mild pretreatment gastrointestinal impairment existed at study entry and might have had an effect on occurrence and severity of AE. Not all AE were considered to be attributable to the study drug; however, differentiation between drug-associated and drug-unrelated toxicosis is difficult, and therefore all AE were listed regardless of suspected cause. This toxicity profile resembles the one in human patients, but in contrast to humans, no peripheral neuropathy was noted after up to 7 patupilone administrations.[30] Pathogenesis of neuropathy in chemotherapeutics that target microtubules is not entirely clear, but can be explained by the disturbance of microtubule function and interference of the axonal transport.[34] Although no neurological AE were noted in this study with a median of 3 patupilone administrations, mild clinical signs might have been overlooked, and cumulative toxicosis could occur.

Seventy-five percent of the dogs received either steroids or NSAIDs. These drugs have a possible antitumor effect in certain tumor types, such as steroids in malignant lymphoma and NSAIDs in transitional cell carcinoma of the bladder and prostate carcinoma.[35, 36] Most dogs experienced disease progression while under these drugs before study entry. However, in 4 dogs, these substances were prescribed shortly before inclusion into the study. A possible positive influence on response attributable to these drugs cannot be excluded in these cases.

This study was a dose-escalating trial; therefore, many animals received low dosages of patupilone and most of the dogs were heavily pretreated and suffered from advanced disease state. Therefore, response assessment was not a primary goal of this trial. A limitation of this study was the short observation of response and the lack of a defined period of time to count as a response. However, partial remission in tumors that are difficult to treat (prostate and anaplastic mammary carcinoma) was unexpected and was interpreted as promising.

Patupilone is a new antineoplastic agent with a manageable toxicity profile and a promising antitumor response in dogs with spontaneously occurring tumors. As patupilone induces an arrest at the most radiosensitive phase of cell cycle (G2/M), it is an ideal radiosensitizer showing supra-additive efficacy in mice.[2, 23, 32, 33] Furthermore, patupilone possesses antimetastatic and antiangiogenic properties, is able to cross the blood-brain barrier and is not susceptible to p-glycoprotein-overexpression-mediated chemoresistance.[2, 37] This makes it a promising agent for single and combined modality treatment.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We thank Yuko Goto-Koshino, University of Tokyo for the canine malignant lymphoma T-cell line; Erin Dickerson, University of Wisconsin, for the canine hemangiosarcoma cell line; Barbara Rutgen, University of Vienna, for her support with the lymphoma cell line. This study was partially performed using the logistics of the Center for Clinical Studies at the Vetsuisse Faculty, University of Zurich: we thank Marina Meli and Regina Hofmann-Lehmann for their support.

Footnotes
  1. 1

    Ixempra, Bristol-Myers Squibb Company, Princeton, NJ

  2. 2

    Invitrogen, Lucerne, Switzerland

  3. 3

    Invitrogen

  4. 4

    Invitrogen

  5. 5

    Invitrogen

  6. 6

    Invitrogen

  7. 7

    Invitrogen

  8. 8

    Invitrogen

  9. 9

    Bucher Biotec AG, Basel, Switzerland

  10. 10

    Bucher Biotec AG

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References