Safety Evaluation of KP-10 (Metastin 45–54) following once daily intravenous administration for 14 days in Dog.

Abstract

Kisspeptin-10 (previously referred as Metastin 45–54), an active fragment of the endogenous full-length kisspeptin-145, is a potential therapeutic agent for reproductive disorders such as infertility, amenorrhea and pubertal delay. A safety evaluation of KP-10 was conducted in dogs at the doses of 30, 100 and 1000 μg/kg, given once daily intravenously for 14 days with a 14 day recovery period. There were no overt signs of drug related toxicity observed in clinical signs, body weights, food consumption, clinical pathology, histopathology, urinalysis, ECG or respiratory rate. Due to very rapid clearance of the peptide, Luteinizing Hormone (LH) levels were measured as a surrogate marker to demonstrate KP-10 exposure. The LH response reached a maximum concentration at 5 minutes post-dose and remained relatively unchanged for at least 30 minutes after dosing with no gender effect. LH concentrations on Day 1 were generally greater than on Day 14. Vaginal cytology results indicated all dogs were in anestrous throughout the dosing period. There were also no KP-10 related findings observed in recovery animals on Day 29. In conclusion, KP-10 demonstrated favorable safety profile in dog where 1000 μg/kg dose was considered as a No Observed Adverse Effect Level dose when administered IV once daily for 14 days.

INTRODUCTION

Kisspeptin (KP, also known as metastin) is one of a family of RFamide-related peptides that was originally found in the human placenta^1,2. The gene, KISS1, that encodes this peptide is first discovered in 1996 as a metastasis suppressor gene³. The product of the Kiss1 gene is a 145 amino acid peptide from which shorter peptides (kisspeptin 54, 14, 13 and 10) are derived. KP-10 (Metastin 45–54, kisspeptin-1 112–121), a shorter C-terminal peptide, shares similar affinities and functions that is shown to activate Kiss1 receptor^1,2,4. The peptide is now widely accepted as an essential endogenous regulator of the GnRH neuroendocrine system⁵. The peptide stimulates GnRH release through Kiss1 receptor (formerly referred as GPR54, AXOR12, hOT7T75). Mutations in Kiss1 receptors are shown to cause hypogonadotropic hypogonadism in humans and mice^6–8, or precocious puberty⁹. Kiss1 receptor was first isolated from rat brain in 1999¹⁰ and is now widely recognized as a molecular switch for puberty¹¹.

Neurons that express Kiss1/kisspeptin are found consistently in two major cell populations, one located in the arcuate nucleus (infundibular nucleus in humans) and in the preoptic region. Kisspeptin neurons directly innervate and stimulate gonadotrophic releasing hormone (GnRH) neurons, which are part of an important pathway for reproduction regulation. In humans, KP and its receptor showed similar distribution with high levels in placenta, pituitary, pancreas, testis, liver, small intestine and spinal cord^2–4. The ability of KP10 to stimulate GnRH release may provide a role as an investigative tool or therapeutic modality in reproductive and pubertal disorders. Therefore, attention has been given to better understand the biologic role of kisspeptin in human. Thus, to support a clinical evaluation of KP-10, safety study in non-rodent species was conducted following GLP regulations.

Kisspeptin has been studied extensively in various animal species such as mice, rats, monkeys, sheep, horse, hamster, goat, pig, frog, dog and fish^12–15. The results from these studies demonstrated robust luteinizing hormone (LH) response to KP treatment indicating presence of kisspeptin system in these species. In dog, immunoreactive KISS1 was found in the oocytes during all stages of the oestrous cycle, in the granulosa cells during all stages except anoestrus and in the corpus luteum (CL) during dioestrus. KISS1 was absent in the ovaries of pre-pubescent bitches¹⁶. KP-10 and GPR54 are expressed in the canine uterus and trophoblast cells¹⁷. Canine KP-10 (YNWNVFGLRY) differs from human KP-10 (YNWNSFGLRF) at two positions and is a potent kisspeptin that elicits robust LH, FSH and oestradiol responses in anoestrous bitches^14,15. These published results provide evidence for the presence of kisspeptin system in dog and thus dog can be used as a relevant animal species for non-clinical safety evaluation of KP-10. Furthermore, the strong response of KP-10 on LH levels indicate pharmacodynamic marker (LH levels) can be used as a surrogate marker to demonstrate exposure to KP-10 in this study, especially if difficulties arise with measuring KP-10 blood levels. After subcutaneous administration of radiolabeled KP-10 analogue ([D-Tyr-14C]TAK-448) to rats and dogs, the dosed radioactivity was almost completely recovered by 48 and 72 hr in rats and dogs, respectively, and most of the radioactivity was excreted in urine after extensive metabolism in the two species²¹.

MATERIALS AND METHODS

Test Article and Formulation:

The test article, KP-10 (white powder with purity 98.4%), was synthesized at the Peptide/Protein Core Facility of the Massachusetts General Hospital and was stored frozen (−20°C). The control article (vehicle) was sterile normal saline (Baxter International, Lot Number: C717124). KP-10 formulations were prepared by dissolving it in approximately half of the required vehicle, followed by stirring, sonication and filtration prior to adding a sufficient volume of vehicle to achieve the target concentration. Aliquots of each dosing formulation were analyzed for concentration using a validated HPLC method.

Animals

Beagle dogs (Covance, Cumberland, Virginia), 8–12 month of age, 7–10 kg body weight were individually housed and provided with Harlan Teklad Certified Dog Chow once daily. Water from the municipal system was supplied, ad libitum, via water bowl.

Animal care, housing and environmental conditions were according to AAALAC recommendations, requirements stated in the Guide for Care and Use of Laboratory Animals and stated by the US Department of Agriculture through the Animal Welfare Act requirements.

Experimental Design

Forty dogs were randomly divided into four groups (5/sex/group), which included vehicle control, 30, 100 and 1000 μg/kg groups (Table 1). KP-10 or vehicle was given as a single IV bolus dose with dosing volume of 2.6 ml/kg, once daily for 14 consecutive days, after which 3/sex/group were necropsied on Day 15, followed by a 14-day recovery period (2/sex/group) dogs were necropsied on Day 29. Route of administration and the duration of treatment were used to support proposed clinical trial of up to 14 days duration using intravenous route of administration.

Table 1:

Study Design-Effect of KP-10 following once daily intravenous administration for 14 days in dog.

Group	Dose (μg/kg)a	Total Number of Dogsc	Necropsy dogsb
			Day 15	Day 29
Control	0	5M/5F	3	2
Low	30	5M/5F	3	2
Mid	100	5M/5F	3	2
High	1000	5M/5F	3	2

^aDosing volume 2.6 ml/kg

^bEqual number of dogs per sex per group.

^cM=Male; F=Female

Safety Evaluation Endpoints

Clinical Observations

Clinical observations for evidence of toxicity were observed and recorded at least twice (AM and PM) daily during the study.

Body Temperatures

Baseline body temperatures were measured and recorded once during pretest, approximately 24 hr after dose administration on Day 1, and prior to necropsy (Study Days 15 and 29).

Body Weights

Individual animal body weights were recorded for all animals pre-study for group assignment, once weekly during the dosing and recovery periods, the day prior to necropsy (non-fasted weight), and prior to termination (Study Days 15 and 29).

Food Consumption

Daily food consumption was measured quantitatively starting on Day 1 and continuing through the end of the study.

Ophthalmic Examinations

Ophthalmic examinations were conducted once during pre-study on all dogs and prior to scheduled necropsy.

Clinical Pathology

Blood samples were collected once during pretest and on Days 2, 8, 15 (5 dogs/sex/group), and 29 (2 dogs/sex/group). Hematology analysis included: WBC, RBC, hemoglobin, hematocrit, MC, MCH, MCHC, platelets, neutrophils, T-lymphocytes, monocytes, eosinophils, basophiles, lymphocytes, reticulocytes and nucleated RBCs. Clinical chemistry analysis included: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, total bilirubin, direct bilirubin, total protein, albumin, glucose, blood urea nitrogen, creatine, phosphates, creatine kinase, lactate dehydrogenase, sodium, potassium, chloride, globulin, albumin/globulin ratio, indirect bilirubin, prothrombin time, activated partial thromboplastin time and fibrinogen.

Urinalysis

A urine sample was collected overnight on Day 14/15 and Day 28/29. Urinalysis endpoints included; bilirubin, blood, glucose, color and gross appearance, ketones, leukocytes, microscopic examination, nitrites, pH, protein, specific gravity, total volume and urobilinogen.

LH (Luteinizing Hormone) Level Determination

Blood samples (~1 mL) were obtained from each dog pre-dose and at 5, 15, and 30 minutes after dosing on Days 1 and 14. Plasma samples were collected and analyzed using radioimmune assay (RIA). The Canine LH RIA test kit (lot # 035) obtained from MP Biomedicals was used for the analysis.

Vaginal Cytology

Vaginal cytology specimens were collected from all female dogs daily from Day −1 through Day 15 to determine estrus cycle stages during the study.

Electrocardiogram (ECG) and Pulmonary Evaluation

ECG waveform signals and respiratory endpoints (rate and abnormal breathing) were collected from all dogs approximately 30 minutes following dosing on Study Day 13. ECG waveform tracings were qualitatively evaluated for evidence of cardiac dysfunctions such as abnormal heart rate, rhythm, and morphology.

Necropsy/Histopathology

Following 14 days of dosing, 3 dogs/sex/group were terminated (Day 15) and subjected to a complete gross examination. Following a 2-week recovery period (Day 29), the remaining dogs (2 dogs/sex/group) were terminated and subjected to a complete gross examination. Euthanasia for necropsy was performed by administration of an barbiturate overdose followed by exsanguination. The following tissues were collected for histopathology examination: adrenal glands, aorta, bone, femur, bone marrow (sternum, costochondral junction), brain, cecum, colon, duodenum, epididymides, esophagus, eyes, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mandibular, and mesenteric), mammary gland, ovaries, pancreas, parathyroid gland, pituitary gland, prostate gland, salivary gland (mandibular), sciatic nerve, seminal vesicle, skeletal muscle (thigh), skin (ventral abdomen), spinal cord (thoracolumbar), spleen, stomach (forestomach, glandular), testes, thymus, thyroid glands, trachea, urinary bladder, and uterus.

The tissues were examined, sampled and fixed in 10% neutral-buffered formalin. All fixed tissues from Groups 1 (control) and 4 (high dose) were trimmed, embedded in paraffin, sectioned at approximately 5 microns, mounted on glass slides and stained with hematoxylin and eosin for microscopic evaluation.

RESULTS

Dose formulation concentration analysis was performed on each newly formulated dose solution. Results of dose formulation analysis confirmed dose concentrations were within the acceptable range (Table 2).

Table 2.

Results of concentration analysis of the dosing formulations

Sample	Target Concentration (μg/mL)	Average Concentration (μg/mL)	% RSDa	Average % REb
Control	0	BLOQc	NAd	NA
Low	12	10.8	2.0	−10.0
Mid	40	38.2	0.5	−4.5
High	400	359	1.0	−10.4

^aRelative standard deviation.

^bRelative error.

^cBelow the limit of quantitation (5 μg/mL).

^dnot applicable

KP-10 treatment did not induce any significant clinical signs, body temperature, body weights, changes in food consumption, clinical pathology, histopathology or respiratory changes that could be attributed to the treatment. There were also no toxicologically significant findings observed during the 14 days of observation period following the treatment.

We evaluated ECG waveform tracings collected from all dogs for approximately 30 minutes following 13 daily doses of KP-10 for evidence of cardiac dysfunction such as abnormal rate, rhythm, and morphology. All dogs maintained sinus rhythms throughout the recording period and were within limits of normal.

We used LH blood levels as a surrogate marker to confirm KP-10 exposure. LH data collected on day 1 and 14 of KP10 treatment are shown in Figures 1 and 2. Control sample was within the acceptable range (5.9 and 9.1 ng/mL) with concentrations of 7.42 and 7.90 ng/mL on both days. Control group and pre-dose treated group LH levels, with a few exceptions, were generally below the limit of quantitation (BLOQ; <2.7 ng/mL). On Day 1, the male and female group mean LH concentrations for the treatment groups were increased, though not dose dependent, above the control group levels at 5, 15, and 30 minutes after dosing, indicating exposure to the test article (Figure 1). In general, the treatment-related response reached a maximum concentration at the 5-minute time point and this response remained relatively unchanged for 30 minutes after dosing, which was the last collection time point. A previous range-finding study in dog showed LH levels drop to baseline levels by 2 hr post-dose (unpublished data). The male and female treated groups had similar profiles for the low and mid dose groups but the male high dose group had higher LH concentrations than the females over the time period tested. On Day 14, the results were similar to the Day 1 results with regard to the treatment group effects relative to control, the absence of a dose-response, the onset and duration of effect, and the similarity between sexes (Figure 2).

Figure 1.

Day 1 LH Concentration Time Curves (Mean ± SEM, sample size=5) for Males and Females following KP10 treatment.

Figure 2.

Day 14 LH Concentration Time Curves (Mean ± SEM, sample size=5) for Males and Females following KP 10 treatment.

Vaginal cytology was performed to track the stage of the estrous cycle of each female in this study. Vaginal cytologies confirmed all female dogs on the study were in anestrous before dosing started and remained in anestrous until dosing was over.

Test article related changes in organ weights (adrenal gland, brain, epididymides, heart, kidney, liver, lung, ovaries, spleen, testes, thymus, thyroid and uterus) were not observed. A complete histopathological evaluation was performed on Groups 1 (control) and 4 (high dose) for identification of target tissues. Histopathologic changes were not identified, therefore, no other dose groups or recovery group were evaluated.

DISCUSSION

The study was conducted to determine the target organ toxicity, reversibility and toxicokinetics of daily bolus intravenous injections of KP-10 for 14 days followed by a 14-day observation period in a non-rodent species as a prerequisite to entering human clinical trials. The doses were selected to include and exceed the proposed therapeutic dose (0.24 nmol/kg, 0.313 μg/kg) in human to provide adequate safety margin.

KP-10 treatment did not induce any significant clinical signs, body temperature, body weights, changes in food consumption, clinical pathology, ECG, respiratory changes or organ weights that could be attributed to the treatment. There were also no toxicologically significant findings observed during the 14 day observation period following the treatment.

There were no drug-related microscopic findings (target organs) in animals of the high dose group on Day 15 necropsy. Therefore, tissues from lower dose groups and recovery groups (Day 29) were not examined microscopically. The significantly lower mean testis weight was accounted for by one smaller testis in one of three dogs, which was attributed to immaturity, and not to the test article. The decreased testis weight correlated the microscopic finding of small seminiferous tubules, and hypo-spermatogenesis. These testicular findings were not considered test article related and were consistent with immaturity in young dogs.²⁴ Furthermore, the affected high dose male did not have the highest LH levels indicating testicular effects were unlikely related to the test article. Thompson et al., (2006) found that the continuous subcutaneous administration of kisspeptin-54/day for 13 days using Alzet minipump in rat cause a decrease in testicular weight, degeneration of the seminiferous tubules and a decrease in the circulating levels of the testes-derived hormones, inhibin B and testosterone. Unlike the rat study¹⁸, pronounced effects on testes were not seen in this study which could be explained by bolus administration as opposed to continuous administration in the rat.

In addition to Kisspeptins in the endocrine system, co-localization of KP and its receptor (GPR54) were also found in the aorta, coronary artery and umbilical vein^11,19. It was demonstrated ex-vivo that KPs are potent vasoconstrictors¹⁹. However, Nijher et al., (2010) did not find any effects of kisspeptin-54 on heart rate or blood pressure for 4 hr post-administration in healthy volunteers²⁰. We evaluated ECG waveform tracings collected from all dogs for approximately 30 minutes following 13 daily doses, anticipating that an effect on ECG waveform tracings would be maximal at the end of dosing phase of KP-10 for evidence of cardiac dysfunction such as abnormal rate, rhythm, and morphology, however, hemodynamic parameters such as blood pressure was not monitored in this study. All dogs maintained sinus rhythms throughout the recording period and were within limits of normal. There were frequent changes in heart rates but these did not appear to be related to the test article and could be due to handling, lights on/off, feeding time, etc. In addition, we did not observe any KP-10 related cardiovascular (blood pressure, heart rate, ECG intervals) or pulmonary (tidal volume or minute volume) effects at 30, 100 or 1000 μg/kg doses when evaluated in a stand-alone single dose safety pharmacology study in telemetered male beagle dogs (unpublished data). This difference between ex-vivo and in-vivo vasoconstrictive effect of KP may be accounted for by the doses, metabolism, clearance, different analog of KP, etc.

After subcutaneous administration of [D-Tyr-14C]TAK-448 to rats and dogs, the dosed radioactivity was completely recovered within 72 hr and mainly excreted in urine after extensive metabolism in dog²¹. Thus, it is difficult to detect and monitor KP-10 blood levels due to its rapid metabolism and the bioanalytical sensitivity needed at low doses. As an alternative, we chose to measure LH blood levels as a surrogate marker to confirm KP-10 exposure. Control sample was within the acceptable range (5.9 and 9.1 ng/mL) with concentrations of 7.42 and 7.90 ng/mL on both days. Control group and pre-dose treated group LH levels, with a few exceptions, were generally below the limit of quantitation (BLOQ; <2.7 ng/mL). On Day 1, the male and female group mean LH concentrations for the treatment groups were increased, though not dose dependent, above the control group levels at 5, 15, and 30 minutes after dosing, indicating exposure to the test article (Figure 1). In general, the treatment-related response reached a maximum concentration at the 5-minute time point and this response remained relatively unchanged for 30 minutes after dosing, which was the last collection time point. A previous study showed LH levels drop to baseline levels by 2 hr post-dose (unpublished data). The male and female treated groups had similar profiles for the low and mid dose groups but the male high dose group had higher LH concentrations than the females over the time period tested. On Day 14, the results were similar to the Day 1 results with regard to the treatment group effects relative to control, the absence of a dose-response, the onset and duration of effect, and the similarity between sexes (Figure 2). A comparison of the Day 1 and Day 14 results, suggested that the LH response was generally greater on Day 1 than Day 14 for a given dose group indicating potential for developing tolerance to the chronic stimulation by KP-10 or receptor saturation or desensitization/down regulation or could likely be due to feedback loop inhibition of overstimulation of KP10 on the Hypothalamic-pituitary-gonadal axis.²⁵

In a previous range-finding study (unpublished data), female dogs did not induce LH in response to the KP-10 treatment. It is reported that the LH response to KP-10 is dependent on estrous cycle where the LH response to KP-10 was most pronounced during anestrus and least pronounced during the first half of the luteal phase¹⁵ It is possible that these female dogs could have been in estrous which may have inhibited LH release in response to the treatment. Therefore, vaginal cytology was performed to track the stage of the estrous cycle of each female in this study. Vaginal cytology confirmed all female dogs on the study were in anestrous before dosing started and remained in anestrous until dosing was over. LH release was observed in both sexes indicating the stage of estrous may be playing a critical role in KP’s activity. Therefore, hormone levels in clinical patients may affect KP-10 activity and, depending on the indication, may be useful in patient selection. Similar effects of KP-10 on LH release was observed in agonadal juvenile monkeys when treated via intracerebral or intravenous single injection to induce a robust discharge of LH within 30 min post injections²². With a low-toxicity non-clinical safety profile in rat (NOAEL > 3000 μg/kg dose, unpublished data) and dog (NOAEL 1000 μg/kg), the KP-10 was tested in healthy adult men, demonstrating that a single, short-lived dose of kisspeptin induced sustained GnRH release as indicated by the kisspeptin-induced LH pulse²³.

The no-observed-adverse-effect-level (NOAEL) for KP-10 was 1000 μg/kg (a human equivalent dose of approximately 500 μg/kg based on body surface area) in dog when administered IV once daily for 14 days, which demonstrated favorable safety profile with more than adequate safety margin for the proposed human clinical dose (0.313 μg/kg).