Effects of Plasmid-Mediated Growth Hormone-Releasing Hormone in ...

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Effects of Plasmid-Mediated Growth Hormone-Releasing Hormone in Severely Debilitated Dogs with Cancer Ruxandra Draghia-Akli,1,2,* Kevin A. Hahn,3 Glen K. King,3 Kathleen K. Cummings,1,2 and Robert H. Carpenter1 1 ADViSYS, Inc., The Woodlands, Texas 77381 Baylor College of Medicine, Houston, Texas 77030 3 Gulf Coast Veterinary Oncology, Houston, Texas 77027 2

*To whom correspondence and reprint requests should be addressed at ADViSYS, Inc., 2700 Research Forest Drive, Suite 180, The Woodlands, TX 77381. Fax: (281) 296-7333. E-mail: [email protected] or [email protected].

Cachexia is a common manifestation of late stage malignancy and is characterized by anemia, anorexia, muscle wasting, loss of adipose tissue, and fatigue. Although cachexia is disabling and can diminish the life expectancy of cancer patients, there are still no effective therapies for this condition. We have examined the feasibility of using a myogenic plasmid to express growth hormone-releasing hormone (GHRH) in severely debilitated companion dogs with naturally occurring tumors. At a median of 16 days after intramuscular delivery of the plasmid, serum concentrations of insulin-like growth factor I (IGF-I), a measure of GHRH activity, were increased in 12 of 16 dogs (P < 0.01). These increases ranged from 21 to 120% (median, 49%) of the pretreatment values and were generally sustained or higher on the final evaluation. Anemia resolved posttreatment, as indicated by significant increases in mean red blood cell count, hematocrit, and hemoglobin concentrations, and there was also a significant rise in the percentage of circulating lymphocytes. Treated dogs maintained their weights over the 56-day study and did not show any adverse effects from the GHRH gene transfer. We conclude that intramuscular injection of a GHRH-expressing plasmid is both safe and capable of stimulating the release of growth hormone and IGF-I in large animals. The observed anabolic responses to a single dose of this therapy might be beneficial in patients with cancer-associated anemia and cachexia. Key Words: dog; GHRH; GH; IGF-I; cancer; cachexia; plasmid; electroporation.

INTRODUCTION Cachexia, generally defined by anemia, weight loss, anorexia, muscle wasting, loss of adipose tissue, and fatigue, is a frequent complication of cancer and other chronic diseases. Its development in cancer patients often precludes further therapy and may contribute directly to death [1,2]. Patients with cachexia are catabolic, showing an increased breakdown of proteins, decreased protein synthesis, a negative nitrogen balance, and an increased basal energy expenditure. These effects may be exacerbated by an inadequate food intake and reduced absorption of food. The release of cytokines such as tumor necrosis factor ␣ (TNF-␣) and interleukins 1 and 6 from the tumor or from normal host cells responding to the tumor further contribute to this condition. Preclinical studies in rodents have suggested that anabolic hormones, such as growth hormone, insulin-like growth factor I (IGF-I), and IGF binding protein 3, may reverse the catabolic state associated with cachexia in

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cancer patients [3,4], but for logistical reasons this strategy has not been tested in large animals with naturally occurring tumors. Of these anabolic agents, growth hormone is of particular interest because of its ability to increase weight, lean body mass, and work output in patients with cachexia due to acquired immunodeficiency syndrome (AIDS) [5]. Although administration of exogenous recombinant growth hormone produces anabolic effects in a variety of situations [6 – 8], therapy with this protein has certain disadvantages. It must be administered subcutaneously or intramuscularly as frequently as once a day over the entire treatment period, and the nonphysiological hormonal peaks and troughs that follow such injections often result in impaired glucose tolerance and insulin resistance [9]. Moreover, biological responses to exogenous growth hormone are not similar to physiologic responses to the naturally occurring isoforms of this protein [10]. Growth hormone synthesis and secretion from the

MOLECULAR THERAPY Vol. 6, No. 6, December 2002 Copyright © The American Society of Gene Therapy 1525-0016/02 $35.00

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anterior pituitary are stimulated by growth hormone-releasing hormone (GHRH), a hypothalamic peptide hormone [11], and low levels of the peptide (approx 100 pg/ml) are sufficient to induce growth hormone secretion [12,13]. Hence, an alternative method to increase growth hormone release would be to administer GHRH. Several studies have shown that continuous infusion of GHRH ensures the production and release of all growth hormone isoforms in their normal pulsatile pattern while maintaining appropriate physiologic feedback mechanisms [14 – 16], thus avoiding adverse effects [17,18]. Unfortunately, the short half-life of the GHRH peptide in plasma (⬍12 min) mandates frequent intravenous or subcutaneous injections of the hormone to sustain its activity [19,20], making recombinant GHRH therapy impractical for chronic conditions. Administration of a DNA plasmid encoding for GHRH could in principle overcome this limitation; indeed, preliminary studies in small animal models suggest that a single injection of a GHRH plasmid into skeletal muscle will ensure physiologic GHRH expression for several months [21]. To test whether this approach to growth hormonereleasing hormone therapy would be feasible and potentially produce beneficial effects in cancer patients, we used a plasmid encoding GHRH to induce both growth hormone and IGF-I synthesis and release in a pilot phase I study in severely debilitated companion dogs with naturally occurring tumors. Follow-up evaluation demonstrated increased serum IGF-I concentrations, an indicator of GHRH activity, together with correction of anemia, weight stabilization, and a significant increase in circulating lymphocytes. These results suggest a role for plasmidmediated GHRH therapy in reversing the catabolic processes associated with cancer cachexia.

RESULTS Forty companion dogs with various spontaneously occurring malignancies were enrolled in the study from January 2001 to December 2001. All were severely debilitated, as judged from owner report of weight loss, poor appetite, muscle weakness, and lethargy and from red cell counts that were below the range in normal healthy dogs. All dogs received standardized chemotherapy, radiotherapy, or combination chemotherapy/radiotherapy of Vincristine 0.6 – 0.7 mg/m2 ⫹ Cytoxan 200 –300 mg/m2 ⫾ Adriamycin 30 mg/m2. Radiotherapy regimen was 80 rad to the local tumor for 16 –21 visits. Twenty dogs were randomized to be injected once in the semitendinosus muscle with the GHRH plasmid (100 ␮g/kg to no more than 1 mg), while the remaining 20 served as untreated controls. Plasmid uptake was enhanced by square-wave pulse electroporation. Four of the treated dogs and one of the control dogs died or were euthanized at the owner’s request within the first 3 days of plasmid injection. Thus, 16 experimental dogs and 19 controls completed the

MOLECULAR THERAPY Vol. 6, No. 6, December 2002 Copyright © The American Society of Gene Therapy

TABLE 1: Presenting feature of 35 debilitated dogs with cancer Dog

Breed

Tumor type

Current therapy

Age (years)

T1

Sheltie

Carcinoma

RT

11

T2

Sheltie

Carcinoma

RT

9

T3

Irish setter

Lymphoma

CT

14

T4

Golden retriever

Lymphoma

CT

14

T5

German shepard

Carcinoma

CT

12

T6

Cocker spaniel

Lymphoma

CT

13

T7

Golden retriever

Sarcoma

CT

13

T8

Chihuahua

Sarcoma

RT/CT

4

T9

Miniature schnauzer

Melanoma

RT/CT

8

T10

Whippet

Sarcoma

RT/CT

11

T11

Scottish terrier

Carcinoma

RT/CT

10

T12

Great Pyrenees

Sarcoma

RT

10

T13

Labrador retriever

Sarcoma

CT

14

T14

Labrador retriever

Carcinoma

CT

9

T15

Bassett hound

Sarcoma

CT

12

T16

Mix breed

Hemangiopericytoma

RT/CT

C1

Mix breed

Carcinoma

RT

9

C2

Pug

Mast cell tumor

CT

10

C3

Mix breed

Sarcoma

CT

10

C4

Pekinese

Melanoma

CT

13

C5

Labrador retriever

Lymphoma

CT

7

C6

Miniature schnauzer

Lymphoma

CT

11

C7

Welsh corgi

Lymphoma

CT

12

C8

Scottish terrier

Lymphoma

CT

9

C9

Rottweiler

Lymphoma

CT

8

C10

Golden retriever

Sarcoma

CT

9

C11

Bulldog

Leukemia

CT

4

C12

Rottweiler

Sarcoma

CT

9

C13

Mix breed

Lymphoma

CT

9

C14

Golden retriever

Sarcoma

CT

7

C15

Golden retriever

Lymphoma

CT

12

C16

Airedale terrier

Sarcoma

CT

10

C17

Labrador retriever

Mast cell tumor

RT/CT

13

C18

Labrador retriever

Sarcoma

RT/CT

9

C19

Golden retriever

Melanoma

RT/CT

12

12

Abbreviations: T, treated; C, control; RT, radiotherapy; CT, chemotherapy.

planned baseline measurements and had at least one blood test during the 56-day follow-up period. The presenting features of these two groups, including tumor type and prior therapy, are reported in Table 1. All dogs survived for the duration of the study.

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FIG. 1. Serum IGF-I levels in dogs treated with plasmid-mediated GHRH therapy. The results are presented as (A) individual values and (B) means ⫾ SEM. The order of data presentation in A corresponds with the listing of dogs in Table 1.

An increase in serum IGF-I concentration over the baseline value was taken as a measure of plasmid GHRH activity [22,23]. On the first posttreatment blood test, 12 of the 16 dogs had increases of 21 to 120% in serum IGF-I, compared with none of the case-matched controls (Fig. 1A; P ⬍ 0.01). Mean (⫾SE) serum IGF-I concentrations in treated dogs before plasmid injection and on the two follow-up evaluations were 37.9 ⫾ 13.1, 55.4 ⫾ 20.4, and 61.4 ⫾ 15.5 ng/ml, respectively (Fig. 1B). Control dogs had IGF-I levels of 35 ⫾ 15 ng/ml on each blood test. Mean (⫾SE) red blood cell (RBC) counts were significantly depressed in these dogs, compared with results in normal healthy dogs: 5.8 ⫾ 0.24 ⫻ 106/␮l vs 6.9 ⫾ 0.08 ⫻ 106/␮l (n ⫽ 28), P ⬍ 0.001. As shown in Fig, 2A, administration of the GHRH plasmid was associated with significant increases in RBC count on both the first and the second evaluations (6.3 ⫾ 0.3 ⫻ 106/␮l vs 5.8 ⫾ 0.24 ⫻ 106/␮l on day 0, P ⬍ 0.005, and 6.3 ⫾ 0.43 ⫻ 106/␮l vs 5.8 ⫾ 0.24 ⫻ 106/␮l on day 0, P ⬍ 0.03, respectively). By contrast, the mean RBC count in control dogs declined by 7.74% (P ⬍ 0.006) on the first blood test and by 12% on the second (P ⬍ 0.02, data not shown). Similar results were obtained for hemoglobin level and hematocrit (Figs. 2B and 2C). We also compared the mean (⫾SE) percentages of circulating lymphocytes before and after plasmid injection, demonstrating a significant posttreatment increase (19.1 ⫾ 4.6% on day 9 –27 vs 10.8 ⫾ 1.8% on day 0, P ⬍ 0.05). Control animals had a statistically nonsignificant decreased percentage of circulating lymphocytes over the same period (17.7 ⫾ 5.1% vs 18.4 ⫾ 3.8%, P ⫽ 0.32). Serum TNF-␣ levels were measured in all dogs both before and after treatment, as well as in healthy normal dogs. Of the experimentally treated dogs, three had detectable baseline TNF-␣ levels that had decreased by 40% at 56 days posttreatment (P ⬍ 0.03). TNF-␣ levels were un-

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changed in all other dogs tested. Total protein levels in serum were not significantly increased in treated dogs. The results of complete blood counts, serum biochemistry determinations, and urinalysis remained within normal reference ranges in both treated and control dogs. Dogs that received the GHRH plasmid had stable weights over the course of the study, (⫾0.5% mean deviation from the baseline measurements), while the controls lost a mean 1.5% of their pretreatment weight. There were no reports of local or systemic adverse effects from plasmid-mediated GHRH therapy by either the investigators or the owners of the dogs. All respondents to a questionnaire administered before and after treatment noted improvement in their dogs’ energy, alertness, appetite, and general well-being.

DISCUSSION Cachexia may directly or indirectly shorten the survival of cancer patients. To test the safety and potential therapeutic activity of plasmid-expressed GHRH in this complication, we administered the plasmid once intramuscularly to severely debilitated, anemic dogs with naturally occurring tumors. Most (75%) of the animals showed a physiological increase in serum IGF-I concentration, which was associated with the correction of anemia and a significant increase in circulating lymphocytes. Moreover, the three treated dogs with detectable baseline serum TNF-␣ levels showed a decrease in these measurements by the end of the study. There were no discernible adverse effects associated with this therapy. Taken together, these observations demonstrate the feasibility of GHRH plasmid therapy to stimulate growth hormone synthesis and release in large animals, leading to the correction of anemia and other catabolic processes associated with cancer cachexia.


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FIG. 2. Hematological values in plasmid-treated versus control dogs. (A) Red blood cell count, (B) hemoglobin concentration, and (C) hematocrit. Values are reported as means ⫾ SEM. Asterisks indicate a significant difference from the baseline (day 0) value at the P ⬍ 0.05 level, while open circles denote a difference from the corresponding control value at the P ⬍ 0.05 level.

Studying the effects of GHRH in companion dogs with naturally occurring cancer has many advantages over more conventional models. In particular, both the animal population and the tumors are genetically heterogeneous, as in randomly selected groups of cancer patients, but in contrast to rodents with transplantable tumors or to large inbred animals with chemically induced tumors. Another benefit is that the plasmid doses given to dogs are comparable to those that would be used in patients. An obvious limitation of this type of study is that the dogs cannot be as intensively or invasively monitored as laboratory animals. For example, long-term weight measurement is precluded by surgical procedures such as limb amputation or tumor resection. Nonetheless, sufficient hematological, biochemical, and clinical data were available in this study to conclude that our GHRH plasmid therapy was both feasible and beneficial in dogs and might be similarly effective in cancer patients with cachexia. Following direct intramuscular injection of the plasmid, we used electroporation to enhance the plasmid’s uptake by skeletal muscle cells. This treatment is mild, requires only low plasmid quantities per kilogram of body weight to be effective [24], and has been widely and


successfully used to transfer plasmids into animals [25– 28] and to facilitate delivery of the anti-tumor drug bleomycin to human tumors [29]. Neither the investigators in this study nor the dogs’ owners reported any distress associated with the use of this technique. Some of the therapeutic benefits of GHRH expression in this study could have been predicted from the known effects of the GHRH/GH/IGF-I axis in preclinical models and from the benefits of growth hormone in patients with AIDS cachexia [5]. However, the corrective effect on hemoglobin level and red cell number was not anticipated, since administration of growth hormone itself to normal beagle dogs over 14 weeks produced a dose-related normochromic, normocytic, and nonregenerative anemia [30]. GHRH stimulates IGF-I, and there are data to suggest that IGF-I rather than erythropoietin is the primary mediator of erythropoiesis during catabolic states and in uremic patients [31] and can induce a proportional increase in body mass and oxygen transport capacity [32]. The increase in hemoglobin, hematocrit, and red blood cell count confirms the in vivo erythropoietic growthpromoting effects of GH that are also observed during GH treatment in GH-deficient children or adults [33,34]. Of

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note, none of these dogs showed evidence of polycythemia following plasmid administration, indicating that this approach to stimulating the GHRH/GH/IGF-I axis restores normal physiological levels of hemoglobin and red blood cell synthesis. Indeed, one experimental dog (T5) with normal hematological findings prior to treatment showed no change in blood values over the study course. The distinctive effects of growth hormone versus plasmid-derived GHRH may indicate the different consequences of stimulating the IGF-I system with physiologic “pulses” of growth hormones as opposed to the large peaks and troughs produced by direct injection of the protein. This hypothesis is supported by the lack of biochemical abnormalities in our GHRH-stimulated animals, including especially the hypoglycemia/insulin resistance observed in recipients of excessive levels of recombinant growth hormone [35]. Thus, plasmid-derived GHRH appears to have the characteristics of the naturally occurring peptide, which would avoid the undesirable effects of injecting recombinant growth hormone. The major concern over stimulating the GHRH/GH/ IGF-I pathway with GHRH to treat cancer cachexia is that activation of this pathway might also enhance tumor cell growth. Indeed, recombinant growth hormone is currently labeled “not for use in patients with cancer.” This restriction was based on the following considerations: (a) a proportion of tumors have receptors for GHRH/GH/IGFI/II, (b) levels of IGF-I are elevated in some patients with cancer, and (c) synthetic GHRH antagonists inhibit growth of transplanted human tumor cell lines in nude mice. However, the current understanding is (a) that the receptors for GHRH on tumor cells are mutated and have a low binding affinity for native GHRH [36 –38], (b) that the elevated levels of IGF-I detected in some patients with cancer may actually originate from the tumor itself, therefore representing a consequence rather than a cause of the tumor [39], and (c) that synthetic GHRH antagonists act not through inhibition of the bioactivity of endogenous native GHRH, but primarily by inhibition of autocrine production of GHRH by tumor cells themselves [37,36]. In fact, there is accumulating evidence to suggest that the GHRH/GH/IGF-I pathway makes little contribution to cancer induction or growth. For example, patients with acromegaly do not have an increased incidence of extracranial cancers, and those who do develop cancer have not been found to have accelerated progress of their disease or shorter survival [40]. Second, epidemiological studies of recipients of native or recombinant growth hormone have failed to show convincingly increased risks of cancer due to growth hormone administration [41,42], with the possible exception of patients with breast and colon cancer. Third, patients with AIDS who received growth hormone did not show an increased risk of development or rates of progression of Kaposi’s sarcoma [5]. Finally, study of IGF-I/IGF-I-binding protein levels in patients with cancer has not revealed any causal or prognos-

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tic relationships [43]. These observations have led to the proposal that the presence of cancer can no longer be considered a contraindication to growth hormone administration [44,45]. Certainly, in our study in dogs, there was no evidence that administration of the GHRH plasmid was associated with accelerated tumor growth. On the contrary, the beneficial effects on the hematological, immunologic, and nutritional status of the dogs allowed them to receive additional and/or higher doses of chemotherapy with the potential to reduce tumor size. This proof-of-principle pilot phase I toxicology and feasibility study addresses a major problem in oncology practice, namely that of cancer-associated cachexia, and proposes a means by which this disability might be reversed. The anabolic effects that we observed following injection of a GHRH plasmid in severely debilitated dogs with cancer would be predicted to be of value in cachectic cancer patients.

MATERIALS

AND

METHODS

DNA constructs. The plasmid pSPc5-12 contains a 360-bp SacI/BamHI fragment of the SPc5-12 synthetic promoter [46] in the SacI/BamHI sites of a pSK-GHRH backbone [47]. The mutated porcine GHRH cDNA was obtained by site-directed mutagenesis of human GHRH cDNA (Altered Sites II in Vitro Mutagenesis System; Promega, Madison, WI), and cloned into the BamHI/HindIII sites of pSK-GHRH. The GHRH cDNA is followed by the 3⬘ untranslated region of growth hormone. Characterization of the vector and the long half-life mutant HV-GHRH was previously described [28]. Animal studies. To be eligible for this study, dogs had to have an expected survival time of at least 56 days following plasmid injection, regardless of their cancer type. This time interval was needed to allow for activation and expression of GHRH from the skeletal muscle and to observe dogs for the clinical effects of growth hormone. The dogs were weighed and bled before plasmid injection. Although the protocol specified measurements on day 0 (baseline) and posttreatment days 14, 28, and 56, the owners’ compliance with the schedule was sporadic, so that most dogs had only two posttreatment blood tests: one on day 9 –27 (mean 16.4 for the experimental group and mean 19 for controls) and another on day 28 –56 (mean 39 days for both groups). A complete blood count, serum biochemical profile, and urinalysis were obtained at each interval and analyzed by the same independent laboratory (Antech Diagnostics, Irvine, CA). Subjective evaluation of the dog’s quality of life was based on the owner’s response to a questionnaire completed at each visit. Serum was collected and frozen for subsequent evaluation of IGF-I concentration, a surrogate marker for growth hormone synthesis and release. Contemporary control dogs (those not receiving plasmid but receiving standard cancer treatment) were evaluated in the same manner as plasmid-injected dogs. Intramuscular injection of plasmid DNA. The endotoxin-free plasmid (Qiagen, Inc., Chatsworth, CA) preparation of pSPc5-12-HV-GHRH was diluted in PBS (pH 7.4) to 5 mg/ml. Dogs were injected with the plasmid before any other protocol-specified radiotherapy or chemotherapy. For dogs on chemotherapy, the injection was administered at no fewer than 5 days before or after the medication. No animal was directly irradiated on or around the injection site. The dogs were anesthetized with Propofol (Abbott Laboratories, IL 4 – 8 mg/kg) and injected with plasmid (100 ␮g/kg to a maximum of 1 mg) directly into the semitendinosus muscle, with use of a 3/10 cc insulin syringe and a 29G1/2 needle (Becton–Dickinson, Franklin Lakes, NJ). Two minutes after injection, the injected muscle was electroporated (six pulses, 100 V/cm, 60 ms/pulse) with a BTX T820 electroporator and two-needle electrodes (BTX, Division of Genetronics,


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Inc., CA), as described [48,49]. Animals were allowed to recover for several hours before rejoining their owners. 15.

IGF-I radioimmunoassay. Dog IGF-I was measured by a heterologous human assay (Diagnostic System Laboratories, Webster, TX). The sensitivity limit of the assay was 0.8 ng/ml; the intra- and interassay variations were 3.4 and 4.5%, respectively. TNF-␣ ELISA. Dog TNF-␣ was measured by a modified heterologous human assay (PeliKine Compact Human TNF-␣ ELISA; Research Diagnostics, Inc., Flanders, NJ). Standards in the assay were diluted to 12.4, 8.2, 4.1, 2.25, 0.7, 0.35, and 0 pg/ml. Two separate sets of standards were prepared for each assay. Each serum sample was assayed at 1:1, 1:2, and 1:10 dilution in assay buffer. Plates were read at 450 nm OD. Normal healthy dogs and most cancer dogs had TNF-␣ values that were lower than 0.7 pg/ml, the limit of sensitivity of the assay and lower than in human subjects [50]. Human TNF-␣ was used as a standard in this assay, while the percentage cross-reactivity to the dog serum was not tested but estimated to be more than 50%. Statistical analysis. A Microsoft Excel statistics analysis package was used. Mean values were compared with Student’s t test or ANOVA, with P ⬍ 0.05 taken as the level of statistical significance.

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17. 18.

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20. 21. 22. 23.

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ACKNOWLEDGMENTS We thank the dog owners for their cooperation and participation in this study. The authors particularly thank Dr. Malcolm Brenner for continuous support and useful discussions. We thank our clinical staff at Gulf Coast Veterinary Oncology, Dr. Kimberly Freeman, Dr. Avenelle Turner, Ms. Sherry Stephens, Ms. Aimee Porter, Ms. Shelia Himes, and Ms. Trish Frank for their excellent patient care. We thank Dr. Douglas Kern for his support of this work and Mr. John Gilbert, Ms. Catherine Tone, and Dr. Lou Smith for the critical correction of the manuscript. We acknowledge support for this study from Advisys, Inc. (The Woodlands, TX). RECEIVED FOR PUBLICATION JULY 24, 2002; ACCEPTED OCTOBER 25, 2002

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