Uses of the Novel Small Peptide, KTH-222, in Treating Human Pancreatic Cancer: Evaluation of Different Treatment Regimens using a Mouse Xenograft Model Uses of the Novel Peptide, in Treating Human Pancreatic Cancer: Evaluation of Different Regimens using a

the effects of these peptides on treating cancer in humans has not well-explored, metastases the of ANP and ABSTRACT KTH-222 is a novel, 8-amino acid length peptide derived from atrial natriuretic peptide and a group of related peptides that reduce cancer cell growth. It was previously reported that KTH-222 reduces the rate of tumor growth and prolongs survival in mice implanted with MIA PaCa-2 human pancreatic cancer cells more effectively than gemcitabine when used as primary therapy. The goal of the present study is to explore the effectiveness of KTH-222 for treating pancreatic cancer when used in two other roles: combination primary therapy and salvage therapy. Female athymic nude mice were inoculated subcutaneously in the right flank with MIA-PaCa-2 tumor cells. KTH-222 was administered as part of a combination primary therapy together with gemcitabine and paclitaxel or as a salvage therapy. When used in combination with gemcitabine and paclitaxel as primary therapy, KTH-222 further reduced tumor volume, although it did not extend the interval until terminal tumor volume was reached. When used a salvage therapy, KTH-222 did extend the interval until terminal tumor volume was reached and, in one case, produced a sustained regression in tumor growth. The results of this study, together with earlier results, suggest that KTH-222 could be used in several different ways in the treatment of pancreatic cancer. Small KTH-222, Treatment Mouse Xenograft Model.


Introduction
There is an ongoing need for the discovery of new chemotherapies that improve the treatment of pancreatic cancer [1][2][3][4][5]. This disease is the third leading cause of cancer-related deaths in the United States, with a 5-year survival rate of only 4% to 7% [6]. The two major, standard-of-care treatments for advanced pancreatic cancer are gemcitabine plus protein-bound paclitaxel, and FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin) [5,[7][8][9][10]. While initially very effective, the long-term utilities these treatment regimens are ultimately limited by toxicity and the development of resistance.
A growing body of evidence supports a role for atrial natriuretic peptide (ANP) and a group of peptides related to it by sequence homology [11,12] or by sharing the same precursor protein [13] in the treatment of cancer. These peptides inhibit the proliferation of human pancreatic cancer cells, as well as those from human lung, hepatic, and gastric cancers, in culture [11][12][13][14][15][16][17][18][19]. Certain of these peptides have also been shown to reduce the growth of tumors derived from human pancreatic cells, as well as those from human breast and lung cancer cells, in mouse xenograft models [13,[19][20][21][22].
Although the effects of these peptides on treating cancer in humans has not yet been well-explored, ANP itself has been reported to reduce metastases after curative lung cancer surgery in human clinical studies [23].
The discovery of drugs for the treatment of pancreatic cancer based on the structure of ANP and related peptides has been

ARTICLE INFO ABSTRACT
KTH-222 is a novel, 8-amino acid length peptide derived from atrial natriuretic peptide and a group of related peptides that reduce cancer cell growth. It was previously reported that KTH-222 reduces the rate of tumor growth and prolongs survival in mice implanted with MIA PaCa-2 human pancreatic cancer cells more effectively than gemcitabine when used as primary therapy. The goal of the present study is to explore the effectiveness of KTH-222 for treating pancreatic cancer when used in two other roles: combination primary therapy and salvage therapy. Female athymic nude mice were inoculated subcutaneously in the right flank with MIA-PaCa-2 tumor cells. KTH-222 was administered as part of a combination primary therapy together with gemcitabine and paclitaxel or as a salvage therapy. When used in combination with gemcitabine and paclitaxel as primary therapy, KTH-222 further reduced tumor volume, although it did not extend the interval until terminal tumor volume was reached. When used a salvage therapy, KTH-222 did extend the interval until terminal tumor volume was reached and, in one case, produced a sustained regression in tumor growth. The results of this study, together with earlier results, suggest that KTH-222 could be used in several different ways in the treatment of pancreatic cancer.
difficult since peptides related to ANP through a common precursor, such kaliuretic peptide, long-acting ANP, and vessel dilator peptide, share no obvious sequence homology with ANP or with each other, even though they all inhibit cancer cell growth [13]. More careful analysis, however, reveals that all of the peptides related to ANP that reduce the growth of cancer cells contain a common, 8-amino acid length motif. The version of this motif that was optimized for activity against pancreatic cancer is the peptide, KTH-222 [19].
KTH-222 inhibits the attachment, proliferation, and development of an invasive morphology in cultured human pancreatic tumor cells [19]. The ability of KTH-222 to interfere with tubulin dynamics may be responsible for these effects. Furthermore, in mice xenografted with MIA PaCa-2 human pancreatic cancer cells, KTH-222 reduced the rate of tumor growth and prolonged survival more effectively, and with less evidence of toxicity, than gemcitabine when used was as sole primary therapy [19].
In order to advance KTH-222 into clinical development as a treatment for pancreatic cancer, it is desirable to select the optimal treatment regimen for clinical trials. This report uses a mouse xenograft model system of human pancreatic cancer to evaluate two additional potential treatment regimens for KTH-222: combination primary therapy together with the gemcitabine plus paclitaxel, and salvage therapy following treatment with gemcitabine plus paclitaxel followed by maintenance treatment with sunitinib.

Xenograft Model System
The xenograft studies were performed at Translational Drug Development (TD2, Scottsdale, AZ) using their standard protocol, which was approved by the TD2 institutional review board (Approved Study #TD3651). Female athymic nude mice were received at 4 weeks of age and were acclimated for at least 5 days prior to study initiation. The mice were housed in microisolator cages and maintained under specific, pathogen-free conditions.
The mice were fed Toland Global Diet® 2920x irradiated laboratory animal diet, and autoclaved water was freely available.
All procedures were carried out under the institutional guidelines of Translational Drug Development Institutional Animal Care and Use Committee, which conform to the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health (Protocol Number TD3651). Animals were identified using transponders.
The mice were inoculated subcutaneously in the right flank with 0.1 ml of a 50% media / 50% Matrigel® mixture containing a suspension of 5x106 MIA-PaCa-2 tumor cells. At the time of inoculation, the mice were 5-6 weeks old. Tumor bearing animals were monitored and tumors were measured periodically until they reached a size of approximately 125mm 3 . Tumor width and length diameters were measured using a digital caliper. The measured values were digitally recorded using animal study management software, Study Director. Tumor volumes were calculated utilizing the formula: Tumor volume (mm 3 ) = (a x b2/2) where 'b' is the smallest diameter and 'a' is the largest diameter [24]. On the thirteenth day following inoculation, designated Study Day 1, forty mice with tumor sizes of 129-188 mm 3 were randomized into three groups of ten plus two groups of five (Table I)

Drug Treatments
Combination Primary Therapy: The dosing regimen for the combination primary therapy evaluation is shown at the in Table   I, section A. Gemcitabine (80mg/kg at 10mg/ml, intraperitoneal) and paclitaxel (30mg/kg in 10mg/kg saline, intravenous) were administered together every third day for 4 cycles beginning on Study Day 1 (i.e., on Studies Days 1, 4, 7, 10) (Groups 2 and 3).
In Group 3, KTH-222 was administered during and following the administration of the other drugs, 3 times per week (0.5mg/ kg at 10mg/kg, intravenously) beginning on Study Day 1 and continuing until the termination of the group. Group 1 received vehicle treatments of saline alone and were given at the same times and volumes as the drug treatments. The dose of KTH-222 was determined as previously described [19]. The doses for each of the other drugs were selected by TD2 based on their experience in using these drug combinations in this xenograft model and were chosen to produce the greatest anti-tumor effects without producing druginduced morbidity or mortality.
Salvage Therapy: Two groups received the same gemcitabine plus paclitaxel primary therapy as did Group 2, followed by maintenance therapy with sunitinib (40mg/kg at 10mg/kg, orally; Study Days 13-34; Table 1

Combination Primary Therapy
The

Salvage Therapy
The tumor volumes of animals given gemcitabine plus paclitaxel primary therapy followed by sunitinib maintenance therapy appeared reduced compared to those only treated with the primary therapy (Group 2; Figure 2) but this difference did not achieve statistical significance over the entire course of the experiment (Group 2; two-way ANOVA, p = 0.053). The time to TTV in Group 4 was, however, extended one week beyond that of Group 2 ( Figure 2).
In contrast, the tumor volumes of animals given KTH-222 salvage treatment beginning on study day 41, following the same primary and maintenance therapies (Group 5), were significantly less than those of Group 2 (two-way ANOVA, p < 0.05), and the time to TTV in Group 5 was extended beyond that of Group 2 by two weeks.

Adverse Effects
The only adverse effects seen in animals treated with KTH-
Employing mice xenografted with human pancreatic cancer cells, we previously demonstrated that KHT-222 was more effective than gemcitabine in reducing the growth of tumors when used as the primary therapy [19]. In particular, KTH-222 produced a significantly greater reduction in tumor volume than gemcitabine in more advanced stages of tumor growth. Because KTH-222 displayed little evidence of toxicity in that study, it appeared to be good candidate for use with other anti-cancer agents in roles such as combined primary therapy and salvage therapy [19]. The present report examined the effectiveness of KTH-222 in decreasing the growth of pancreatic cancer cells in these alternative roles using the same mouse xenograft system.
In the combination primary therapy application, KTH-222 increased the effectiveness of gemcitabine plus paclitaxel therapy, a standard of care in pancreatic cancer treatment [5,[7][8][9][10] it may have the same benefits as palliative cancer care [27,28].
Palliative care has been shown to not only increase QOL but also extend median survival in human patients, perhaps through a combination of enhancements in both function and mood [27].
To test the use of KTH-222 as salvage therapy, it was given when tumor growth had resumed after gemcitabine plus paclitaxel primary therapy and sunitinib maintenance therapies had been given. Although sunitinib is not routinely used as maintenance therapy in pancreatic cancer, it been shown to be effective in this role [29]. This, however, does not seem likely since all mice were xenografted from the same culture. It also may have been due to some peculiarity in the physiology of the mouse, but this also seems improbable since all mice were of the same strain. Alternatively, in may be that small differences in injection sites placed the xenografted cells in environments which varied in how supportive they were to tumor growth. The ability of the cells to attach and invade would have been compromised by the interference in tubulin dynamics produced by KTH-222 [19], and this may have been sufficient to produce regression in less favorable environments. In addition, if the growth fraction was reduced by an inhospitable environment, the inhibitory effect of KTH-222 on cell division may also have made it impossible for the tumor to expand [19]. Such heterogeneity in growth environments also occurs with human tumors, particularly those resulting from metastases, raising the possibility that KTH-222 treatment could also produce the regression of certain human tumors [30].
Taken together, the results obtained to date suggest that KTH-222 has the potential to be a useful treatment for pancreatic cancer in a variety of roles. In particular, the results from the current study suggest the utility of KTH-222 in combined primary and salvage therapy treatment regimens. Those of a previous study demonstrated the effectiveness of KTH-222 as a sole primary therapy [19]. Further research be necessary to confirm the best use(s) of KTH-222 in the treatment of pancreatic cancer and, thereby, to position the peptide to enter clinical trials.