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. Author manuscript; available in PMC: 2015 Dec 2.
Published in final edited form as: Cancer. 2015 Apr 1;121(14):2411–2421. doi: 10.1002/cncr.29344

Targeting the Interleukin-11 Receptor α in Metastatic Prostate Cancer: A First-in-man Study

Renata Pasqualini 1,2,b,*,#, Randall E Millikan 1,2,#, Dawn R Christianson 1,2,c, Marina Cardó-Vila 1,2,b, Wouter H P Driessen 1,2,d, Ricardo J Giordano 1,2,e, Amin Hajitou 1,2,f, Anh G Hoang 1,2, Sijin Wen 3,g, Kirstin F Barnhart 4,h, Wallace B Baze 4, Valerie D Marcott 2, David H Hawke 5, Kim-Anh Do 3, Nora M Navone 1, Eleni Efstathiou 1,2, Patricia Troncoso 5, Roy R Lobb 6, Christopher J Logothetis 1,2, Wadih Arap 1,2,b,*
PMCID: PMC4490036  NIHMSID: NIHMS670557  PMID: 25832466

Abstract

BACKGROUND

Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. We have worked systematically to map human endothelial receptors (“vascular ZIP codes”) within tumors through direct peptide library selection in cancer patients. Previously, we selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature.

METHODS

We generated a ligand-directed peptidomimetic drug (Bone Metastasis Targeting Peptidomimetic-11; BMTP-11) for IL-11Rα-based human tumor vascular targeting. Pre-clinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effect, PK/PD, and dose-range determination including formal (GLP) toxicity across rodent and non-human primate species. We also report the initial BMTP-11 clinical development: A single-institution, open-label, first-in-class, first-in-man trial (NCT00872157) in metastatic castrate-resistant prostate cancer patients.

RESULTS

BMTP-11 was pre-clinically promising and was therefore chosen for clinical development in patients. We enrolled a limited number of castrate-resistant prostate cancer patients with osteoblastic bone metastases into a phase zero trial with biology-driven endpoints. We demonstrate biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, we identified the MTD on a weekly schedule (20-30 mg/m2). Finally, we determined a renal DLT, namely, dose-dependent reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine.

CONCLUSIONS

These biological endpoints establish BMTP-11 as a targeted drug candidate in metastatic castrate-resistant prostate cancer. Within a larger discovery context, our findings indicate functional tumor vascular ligand-receptor targeting systems may be found through direct combinatorial selection of peptide libraries in cancer patients.

Keywords: BMTP-11, clinical trial, IL-11 receptor α, prostate cancer, vascular targeting

INTRODUCTION

Several lines of evidence indicate that blood vessels of tumors express unique receptors that act as vascular “ZIP codes” and can be targeted with ligands.1-3 Although the identification of functional ligand-receptors within the tumor vasculature is challenging, we recently showed that screening peptide libraries directly in humans enables unbiased target identification.4-7 Nevertheless, whether these targets can be translated into therapies remains unclear.

Treatment options for metastatic castrate-resistant prostate cancer are limited and development of new therapeutic approaches is urgently needed. We identified a peptide motif (CGRRAGGSC) that binds to the interleukin-11 receptor alpha (IL-11Rα) in the tumor vascular endothelium by administering a phage library to a brain-dead cancer patient.4-6 Human IL-11Rα is overexpressed and had similar expression profiles of IL-11Rα and CD31 co-localization during tumor progression and metastases in a large cohort of prostate cancer patients; specific drug binding to IL-11Rα and dose-dependent apoptosis induction of prostate cancer cells8 is mediated through a receptor-interacting site within IL-11.9 Independent groups confirmed this ligand-receptor system by characterizing the binding of CGRRAGGSC.10,11

Based on these findings, we designed a ligand-directed agent, Bone Metastasis Targeting Peptidomimetic-11 (BMTP-11). BMTP-11 consists of the CGRRAGGSC motif synthesized in tandem to D(KLAKLAK)2, an apoptosis-inducing motif that is active upon cell internalization and validated in pre-clinical models of cancer, obesity, and retinopathies.8,12-17

Here we report pre-clinical studies of BMTP-11 across rodent and non-human primate species, and the phase zero BMTP-11 trial in castrate-resistant prostate cancer patients. This is the first-in-class, first-in-man clinical trial to emerge from our long-standing human mapping project.4-7 Our findings include selective BMTP-11 localization to human bone metastasis and targeted tumor apoptosis induction, providing evidence of drug activity and tumor toxicity in humans. These biological endpoints establish BMTP-11 as a targeted drug candidate against human prostate cancer and support its further development.

MATERIALS AND METHODS

Detailed methods are described in the Supporting Experimental Procedures. Pre-clinical efficacy studies in rodents, pharmacokinetic, safety/toxicology studies in non-human primates, and criteria for patient entry followed procedures described in Supporting Methods.

RESULTS

Pre-Clinical Efficacy of BMTP-11 Against Prostate Cancer Xenografts

To evaluate BMTP-11 efficacy in pre-clinical settings, two classical nude mouse models were chosen: a PSA-producing, androgen-dependent model (LNCaP-derived) and a non-PSA-producing, androgen-independent model (DU145-derived).8 As a third experimental system, we selected the bone-forming prostate cancer MDA-PCa-118b model,18 which forms osteoblastic lesions in SCID mice and uniquely recapitulates human prostate cancer.

In pilot experiments, nude mice bearing DU145-derived tumor xenografts received BMTP-11 (5-15 mg/kg, subcutaneous (SQ), every other day, good manufacturing practice (GMP)) or control (Supporting Fig. S1). BMTP-11 efficacy was assessed by serial changes in tumor volume. Tumors were reduced (P<0.0001) relative to controls in nude mice treated with 10-15 mg/kg (Supporting Fig. S1A), whereas lower BMTP-11 doses were less effective (Supporting Fig. S1B,C).

We next assessed the efficacy of BMTP-11 (10 mg/kg, once weekly) by either intravenous (IV) or SQ administration in DU145- (Fig. 1A-C) or LNCaP-derived (Fig. 1D-F) tumor xenografts. Differences in either DU145- or LNCaP-derived tumor volumes in treated nude mice (P<0.0001) were observed relative to controls and, the optimal dose of BMTP-11 in tumor-bearing mice was determined at ~10 mg/kg/week.

Figure 1.

Figure 1

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BMTP-11 efficacy in tumor-bearing immunodeficient mouse models of prostate cancer. (A) DU145-derived tumor-bearing nu/nu (nude) mice were treated with either BMTP-11 (10 mg/kg, n=10) or control (saline, n=10) intravenous (IV) by tail vein administration once weekly for four weeks. Tumor growth was serially monitored and volumes were measured over time. Data are means ± SEM. (B) In a mixed-effects model, there was a significant difference over time in the average percent change from baseline for tumor volume in the treated versus control animals (P< 0.0001). (C) Tumor volumes on day zero (pre-treatment) and day 28 (post-treatment) are shown. (D) LNCaP-derived tumor-bearing nude mice were treated with either BMTP-11 (15 mg/kg, n=4) or control (saline, n=4) by IV administration three times per week for two weeks. Tumor growth was serially monitored and volumes were measured over time. Data are means ± SEM. (E) In a mixed-effects model, there was a significant difference over time in the average percent change from baseline for tumor volume in the treated versus control animals (P< 0.0001). (F) Tumor volumes on day zero (pre treatment) and day 28 (post treatment) are shown. (G) Expression of interleukin-11 receptor alpha (IL-11Rα) in MDA-PCA-118b implanted tumors (upper left) relative to IgG isotype negative control (upper right). Anti-CD31 (lower left) and hematoxylin and eosin (H&E) stain (lower right). T denotes tumor, * denotes bone. (H) Severe combined immunodeficient (SCID) mice bearing MDA-PCA-118b implanted tumors were divided into equal pre-treatment cohorts using X-ray imaging, and (I) bone to tumor stroma ratio was quantified in each group by the relative mean gray-scale analysis prior to treatment initiation. (J) MDA-PCA-118b tumor-bearing SCID mice were treated with either BMTP-11 (10 mg/kg, n=5) or control (saline, n=5) IV via tail vein once weekly for three weeks. Tumor growth was serially monitored and volumes were measured over time. Data are means ± SEM. (K) In a mixed-effects model, there was a significant difference over time in the average percent change from baseline for tumor volume in the treated versus control animals (p< 0.0001). (L) Tumor volumes on day zero (pre-treatment) and day 21 (post-treatment) are shown.

Marked expression/localization of IL-11Rα was found in MDA-PCa-118b tumors,18 but not in non-malignant stroma (Fig. 1G). Heterogenous IL-11Rα expression substantiated pre-treatment cohort assignment of tumor-bearing SCID mice using bone-to-soft tissue ratio within tumors by quantitative X-ray (Fig. 1H,I). BMTP-11 treatment (10 mg/kg weekly, IV) had anti-tumor effects compared to controls (P<0.0001, Fig. 1J-L). The average control-treated tumors increased by 235% (range, 62-520%); in contrast the change in BMTP-11-treated tumor-bearing SCID mice was only 10.6% (range, -40 to 74.3%), thus indicating nearly complete growth suppression (Fig. 1K).

BMTP-11 Tissue Distribution

To evaluate BMTP-11 tissue distribution polyclonal antibodies (IgGs) against the BMTP-11 proapoptotic domain were produced in rabbits using a single D(KLAKLAK) peptide (Supporting Fig. S2A). The antibody showed anti-BMTP-11 immunoreactivity as well as to two other positive controls (Supporting Fig. S2B), whereas there was no immunoreactivity against a negative control peptide (Supporting Fig. S2C). Affinity-purified anti-D(KLAKLAK) IgGs detected BMTP-11 in a concentration-dependent manner (Supporting Fig. S2D).

BMTP-11 immunoreactivity was observed in non-tumor-bearing mice injected with BMTP-11 (25 mg/kg) in the kidneys 20 minutes post-injection (Supporting Fig. S3A) and restricted to the proximal tubules (Supporting Fig. S3B).

Next, BMTP-11 metabolism was analyzed in tissues using MALDI-TOF MS19 (Supporting Fig. S4). While the absolute concentration of each BMTP-11 metabolite could not be quantified unequivocally, the relative abundance of individually identified metabolites was measured based on peak intensities relative to BMTP-11 and other internal standards. Kidneys from mice injected with BMTP-11 revealed BMTP-11 disappearance correlated strongly with the appearance of GG-D(KLAKLAK)2, G-D(KLAKLAK)2 and D(KLAKLAK)2 metabolites (Supporting Fig. S4B). We found that D(KLAKLAK)2 was the predominant metabolite, with no detectable levels of the parent compound or metabolites containing any portion of CGRRAGGSC 4 hours post-administration (Supporting Fig. S4C). These results indicate that the immunoreactivity in the kidney sections may represent a combination of the drug and its metabolic derivatives.

BMTP-11 Stability and Interaction

We established assays to evaluate BMTP-11 in vitro, in vivo and ex vivo. BMTP-11 stability in saline was determined by mass spectrometry. BMTP-11 was solubilized and incubated at RT or 37°C, and serial aliquots were analyzed. Spectra analyses showed only two major peaks, both corresponding to BMTP-11: the first peak was the single-charged form and the 2nd peak corresponded to the double-charged form (Supporting Fig. S5). The latter peak was not present in samples incubated for 8-24 hours thus, establishing that BMTP-11 is stable in aqueous solutions (Supporting Fig. S5).

Finally, to evaluate the functional attributes of BMTP-11 in a pre-clinical setting, bone marrow supernate from human prostate cancer patients were collected, incubated with the drug, and its activity against prostate cancer cells in vitro was unchanged compared to BMTP-11 that was not pre-treated (Supporting Fig. S6).

BMTP-11 Pharmacokinetics in Rodents

To profile the biodistribution and pharmacokinetic properties of BMTP-11, we first carried out pilot studies in mice using 125I-BMTP-11 (Supporting Fig. S7). Mice received 125I-BMTP-11 (15 mg/kg, IV) or PBS and blood samples were serially collected post-injection. After 24 hours, mice were euthanized, and their organs were collected and analyzed using a gamma-scintillation counter. This iodination strategy determined a specific 125I-BMTP-11 activity of 0.27 mCi/g. Based on a standard curve (Supporting Fig. S7A), we estimated that 0.05% of the dose would constitute a good signal- to-noise ratio (~50-fold). Circulating 125I-labled BMTP-11 levels dropped quickly, and by 15 minutes post-injection, only ~25% of the dose was present in whole blood; radioactivity levels in whole blood no longer decreased after 4 hours (Supporting Fig. S7B). After 24 hours, most of the radioactivity was in the kidneys, liver, spleen, and heart (Supporting Fig. S7C). A pharmacokinetics profile was calculated using the standard curve and a non-compartmental analysis (NCA) was performed (Supporting Table S1 and Supporting Fig. S7D).

BMTP-11 Pharmacokinetics in Non-human Primates

We next assessed the pharmacokinetics of BMTP-11 in cynomolgus monkeys (Fig. 2 and Supporting Fig. S8). We detected BMTP-11 in plasma spiked with drug concentrations as low as 0.003 µg/ml (Supporting Fig. S8A, B). Ratios between BMTP-11 and D(KLAKLAK)2, the internal standard (Supporting Fig. S8C) showed good linearity (Fig. 2A), indicating CGRRAGGSC did not alter the stability of D(KLAKLAK)2. Cynomolgus monkeys received IV infusions of BMTP-11 at 1, 3, or 9 mg/kg over 2 hours to mimic the intended clinical application. Plasma samples were collected over the course of 24 hours to generate plasma concentration-time curves for each animal receiving each dose (Fig. 2B). Our results indicate BMTP-11 levels increased during infusion and decreased exponentially to background levels at 8 hours. Area under the curve (AUC) from zero to infinity was calculated from each plasma concentration-time curve. In the dose ranges evaluated, AUC increased proportionally with increased dose, indicating that BMTP-11 clearance mechanisms were neither saturated (Fig. 2C) nor concentration-dependent. The data were best-fitted to a one-compartment open body model (Fig. 2D), and pharmacokinetic parameters were calculated (Table 1). The elimination rate constant for BMTP-11 was calculated using the terminal portion of the plasma concentration-time curves. The curve corresponding to the lowest BMTP-11 dose tested (1 mg/kg) was not used because samples collected 2 hours after the start of infusion (prior to discontinuing infusion) and 5 minutes post-infusion at this dose were undetectable. Preliminary assessment of BMTP-11 metabolites in plasma from monkeys was determined from their molecular mass (Fig. 2E).

Figure 2.

Figure 2

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BMTP-11 pharmacokinetics and metabolites in non-human primates. (A) BMTP-11 standard curve in the plasma of cynomolgus monkeys. (B) BMTP-11 plasma concentration time curve after IV infusion of BMTP-11 (1, 3 and 9 mg/kg) for two hours. (C) Dose linearity plot for the three different BMTP-11 doses. (D) Observed values and model-prediction fitted to a one-compartment open body model by WinNonlin are shown. (E) Identified metabolites of BMTP-11 in plasma and their predicted amino acid sequence were based on molecular mass. Boxed top sequence indicates the parent drug, BMTP-11 (Peak # 2483). Disulfide bonds are indicated by S-S.

Table 1.

BMTP-11 pharmacokinetic parameters in non-human primates

Estimate ± Standard Error
Parameter Units 3 mg/kg 9 mg/kg
Dose mg 20.1 71.1
AUC μg.hour/ml 8.88 ± 1.04 26.81 ± 2.14
Cl liter/hour 2.26 ± 0.27 2.65 ± 0.21
K10 1/hour 0.78 ± 0.22 1.03 ± 0.22
Half-life hour 0.89 ± 0.25 0.67 ± 0.14
Vd liter 2.90 ± 0.59 2.58 ± 0.45
MRT hour 1.28 ± 0.35 0.97 ± 0.20
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AUC, area under the curve; Cl, clearance; K10, elimination rate constant; Vd, volume of distribution; MRT, mean residence time

Dose Range-determination and Toxicity Studies in Rodents and Non-human Primates

GLP studies on mice, rats, and monkeys were conducted to identify dose-range and administration route to formally assess BMTP-11 safety (Supporting Table S2). Within the non-acute group (defined as greater than 50% of animals surviving to study termination), renal injury, such as hyperplastic and/or regenerative lesions as determined by clinical pathology and/or gross necropsy, was the predominant toxicity, with concentration-dependent severity across rodents and primates. Clinical chemistry parameters related to renal function such as serum creatinine and BUN were often elevated but generally returned to baseline at the end of the study indicating renal adaptive/regenerative response. To rule out a neutralizing effect contributing to transient renal findings, we measured serum levels of anti-BMTP-11 (anti-drug antibodies) from single- and multiple-dose studies in rodents and non-human primates (Supporting Fig. S9). Our results indicate that none of the mice treated with a single-dose (3-10 mg/kg) or monkeys treated with multiple BMTP-11 doses (4 weekly doses at 30 mg/kg/dose) developed IgM or IgG anti-BMTP-11 antibodies; serum samples collected from animals in these studies did not show immunoreactivity against BMTP-11 (Supporting Fig. S9A,B). Because CGRRAGGSC mimics native IL-11 binding to IL-11Rα, which activates STAT3,9 we measured anti-IL-11 serum levels to ascertain whether there was a humoral immunogenic response against BMTP-11. Neither IgM nor IgG was detected above background (Supporting Fig. S9C), indicating that BMTP-11 is unlikely to induce a humoral response. Tumor toxicity results and the lack of an immunogenic response led to a formal definitive GLP safety assessment study of BMTP-11 at doses 1, 3, 6, and 9 mg/kg in non-human primates to complete pre-clinical requirements for clinical trials.

Pre-Clinical Safety of BMTP-11 in Non-Human Primates

A GLP-compliant safety study was carried out using cynomolgus monkeys to mimic the clinical regimen expected (weekly IV for four doses). The aims of this non-clinical safety evaluation in primates were to: (i) identify target organs for toxicity and determine whether toxicity is reversible, (ii) determine starting dose and dose-escalation, and (iii) identify parameters for safety monitoring in humans. A recovery group at the highest dose (9 mg/kg) was included. Clinical signs attributed to BMTP-11 included alopecia, vomiting, dehydration, increased urine output, and erythema. These clinical signs were dose-dependent and observed at the highest doses (6 and 9 mg/kg).

Hematologic findings included mild leukocytosis, anemia and mild thrombocytopenia. Mild-to-marked azotemia was noted one week following the initial BMTP-11 dose. Azotemia lessened with continued administration and was completely resolved at the end of the recovery period, with the exception of two out of three monkeys in the highest-dose group.

Alterations in urinary analytes included glucosuria, proteinuria, leukocyturia, and increased transitional/renal epithelial cells. The magnitude of proteinuria and cell counts in the urine decreased with continued drug administration; most urinary abnormalities resolved by the end of the recovery period.

Primates necropsied 24 hours after the final BMTP-11 infusion, showed dose-dependent findings primarily in monkeys receiving 3-9 mg/kg. Pale discoloration of the kidneys was grossly consistent with nephrosis. Kidney weights increased at the highest BMTP-11 dose. Histologic lesions were identified in the kidneys, stomach, pancreas, and infusion sites. Similar dose-dependent lesions were described as degenerative/necrotic, regenerative/reparative, and fibrotic and were reported for BMTP-78,14 adipotide,17 and other D(KLAKLAK)2-containing peptidomimetics. Tubular necrosis and regeneration were noted in monkeys receiving BMTP-11 at any dose; however, these findings were graded minimal-to-mild at the two lowest dose levels, and mild-to-moderate fibroplasia was observed only at the two highest dose levels.

Additional pre-clinical safety GLP studies were performed using a large cohort (n=50) of primates (rhesus and cynomolgus monkeys) withD (KLAKLAK)2-containing drugs. 14,17 Details on some multiple-dose studies have been reported.17 No lethality resulted from dose-dependent toxicity in monkeys receiving single BMTP-11 doses up to 100 mg/kg. Pre-clinical safety studies identified renal tubules as the primary non-target tissue for adverse events with no irreversible toxicity encountered at the highest repeated doses tested. In the absence of an identified lethal dose, we proposed an allometrically estimated dose of 18 mg/m2 in humans, corresponding to a BMTP-11 dose equivalent to 1.5 mg/kg in cynomolgus monkeys20 as a conservative starting dose for a first-in-man clinical trial in castrate-resistant prostate cancer patients with high-volume osseous metastasis. This starting dose is equivalent to only 5% of the dose level associated with reversible renal toxicity in monkeys.

BMTP-11 Clinical Trial

The first-in-man study # NCT00872157 (http://clinicaltrials.gov/ct2/show/NCT00872157, IND 102736) was designed to document ligand-directed targeting of BMTP-11, and to evaluate a relationship of dose to activity and toxicity that would support a larger dose-selection trial. A patient cohort (n=10) was identified and screened in the trial with “intent-to-treat.” During pre-enrollment screening, four patients were deemed non-eligible for trial entry because of low hemoglobin level (n=1) or absence of tumor metastases upon bone marrow biopsy (n=3). The remaining patients (n=6) were enrolled in dose-escalation cohort and treated with BMTP-11 starting at 18 mg/m2 IV weekly for four doses. All 6 patients enrolled had high-volume, castrate-resistant bone metastases for which no standard therapy options were available (Table 2). Patients had received a median of two previous chemotherapy regimens (range, 1-7), and three (50%) had undergone systemic radionuclide-based therapy. All patients had readily demonstrated biopsy-proven prostate cancer in bone prior to registration, and underwent a repeat biopsy within 2-4 hours after the first BMTP-11 dose. All patients entered in this study had baseline IL-11Rα expression relative to a negative control IgG (Fig. 3).

Table 2.

Baseline patient demographics and clinical features by BMTP-11 dose

18 mg/m2
 36 mg/m2
 27 mg/m2
Pt #1 Pt #2 Pt #3 Pt #4 Pt #5 Pt #6
Age at diagnosis 62 55 63 57 67 63
Age at trial registration 74 69 68 61 68 65
Interval, androgen deprivation to CRPC (years) 5.3 7.0 2.0 0.2 0.4 0.5
Interval, CRPC to trial registration (months) 78 38 36 38 8 12
Number of prior cytotoxic therapies 2 2 5 6 1 2
Previous treatment with abiraterone Yes Yes No Yes No No
Serum PSA at registration 2141 334 384 711 321 53
Serum alkaline phosphatase at trial registration 259 293 520 443 447 388
Hemoglobin at trial registration 11.5 11.9 10.5 8.2 8.9 9.2
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Pt, patient; CRPC, castrate-resistant prostate cancer; PSA, prostate-specific antigen

Figure 3.

Figure 3

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Expression of Il-11Rα and negative control (IgG isotype) in human bone marrow. Each patient of the treated cohort (n = 6) underwent bone marrow biopsy prior to receiving BMTP-11.

Our trial required post-treatment bone metastasis biopsies for BMTP-11 drug localization (Fig. 4A). Patients #1 and #2 were treated at the lowest dose level (18 mg/m2), and each received all four planned BMTP-11 doses with no clinically apparent toxicity despite mild increases in serum creatinine, dipstick proteinuria, and urinary casts. Patient #3 was treated at the highest dose level (36 mg/m2) and had grade 3 decrease in glomerular filtration; serum creatinine decreased from 0.7 mg/dl at baseline to 3.8 mg/dl on day 15; consequently he received only two BMTP-11 doses. The protocol was then modified to require more aggressive post-BMTP-11-forced diuresis, and re-opened afterwards at an intermediate dose level (27 mg/m2). Three patients were subsequently treated at the 27 mg/m2 dose. Patient #4 completed the four planned doses with minimal renal toxicity. Patient #5 went off-study after two doses due to disease progression. On day 15, his serum creatinine increased from a baseline of 0.6 mg/dl to 1.3 mg/dl, and his urine protein increased from 151 to 1,840 mg/24-hour. Patient #6 received three BMTP-11doses, and came off-study on day 22 because his urine protein increased from 132 to 2,180 mg/24-hour.

Figure 4.

Figure 4

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BMTP-11 targets bone metastasis in patients with castrate-resistant prostate cancer. (A) Scheme of the first-in-man clinical trial design. (B) BMTP-11 detection by mass spectrometry (cleaved proapoptotic domain, left; parent BMTP-11 compound, right). (C) BMTP-11 immunocolocalization and (D) TUNEL assay are shown pre- and post-treatment with BMTP-11 (18 mg/m2). Representative patient samples are shown.

A secondary objective was to define acute toxicity in patients. There were no treatment-related deaths and no grade 4 events. All adverse events of grade 2 or above according to the NCI21 are shown regardless of attribution (Table 3). Most of the events (anemia, elevated alkaline phosphatase, and hypoalbuminemia) reflect metastatic prostate cancer affecting bone. Two patients (33%) came off-study for dose-limiting renal toxicity. Consistently, proteinuria and increase in serum creatinine were the most prominent toxicities identified in the formal pre-clinical evaluation of BMTP-11 in rodents and non-human primates.

Table 3.

Adverse events*

Adverse event Grade 3 Grade 2
Proteinuria 0 6
Back pain 0 1
Pain, not otherwise specified 0 2
Low serum albumin 0 1
Alkaline phosphatase 2 2
Anemia 2 2
Constipation 0 1
Creatinine 0 1
Fatigue 0 2
Glomerular filtration rate 1 1
Hyperglycemia 1 0
Hyponatremia 1 0
Hypokalemia 1 0
Joint effusion 0 1
Depression 0 1
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*

Patients with an adverse event of the indicated grade

BMTP-11 localization in treated patients was determined by mass spectrometry analyses (Fig. 4B) and/or immunohistochemistry (Fig. 4C). Notably, in all six patient biopsies (100%; 95% CI, 54-100%), BMTP-11 accumulated at bone metastasis sites, indicating binding to prostate cancer following IV infusion and consistent with the pre-clinical data. Analysis of all tissue samples by TUNEL staining showed tumor-apoptosis and BMTP-11 co-localization (Fig. 4D).

With respect to clinical activity, we observed no responses defined by the PCWG2 criteria.22 Moreover, a heavily pre-treated patient (Patient #4, Table 2) treated at the 27 mg/m2 level showed marked symptomatic improvement and experienced transient simultaneous declines in serum PSA, alkaline phosphatase, and lactate dehydrogenase coincidentally during BMTP-11 treatment, but the tumor progressed rapidly when the candidate drug was discontinued (Fig. 5).

Figure 5.

Figure 5

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Serial changes in serum tumor markers of a patient pre- and post-treatment with BMTP-11. Serum PSA, alkaline phosphatase, and lactate dehydrogenase are shown. Abbreviations used: T/C: paclitaxel plus carboplatin IV every 3 weeks; VP-16/CDDP: etoposide plus cisplatin IV every 3 weeks; KA/VE: ketoconazole PO plus doxorubicin IV alternating on a weekly basis with estramustine PO plus vinblastine IV; DES/Dex: diethylstilbestrol PO plus dexamethasone PO; T/S: docetaxel IV plus sunitinib PO; G/S: gemcitabine IV plus sunitinib PO; CVD: cyclophosphamide IV and vincristine IV, plus dexamethasone PO; 5-FU: infusional 5-fluoro-uracil plus concomitant with external beam radiotherapy.

DISCUSSION

Although the biology of castrate-resistant prostate cancer remains poorly understood, most patients have osteoblastic bone metastases. With the exception of bone-seeking radiopharmaceuticals,23,24 the development of drugs targeting the bone metastasis tumor microenvironment has lagged behind new hormonal agents,25-28 immunotherapy,29 and cytotoxics.30

The pre-clinical evaluation of BMTP-11 activity and its translation into an early clinical application in patients included targeted efficacy, the development of ligand-directed drug detection methodology, and safety/toxicology studies in rodent and primates. All these data were used to design and conduct a first-in-human clinical trial. The primary endpoints of the study in prostate cancer patients were to document physical targeting of BMTP-11, and evaluate the relationship of dose to toxicity and efficacy in humans that would support a full phase I trial. The clinical dose-limiting toxicity was somewhat predictable from our pre-clinical tissue distribution and GLP safety/toxicology studies; weekly BMTP-11 administration in the dose-range studied was associated with renal toxicity. Pre-clinical safety study alterations in serum biochemical and urinary analytes were attributed primarily to nephrotoxicity and altered proximal tubular function, with minimal changes identified in animals receiving the lowest doses. Renal toxicity is attributable to the D-enantiomer pro-apoptotic moiety. Our studies revealed that the targeting moiety (composed of L amino acid residues only) was no longer present in the plasma 4 hours post-injection. Thus, all targeted D(KLAKLAK)2 peptidomimetics present a very similar toxicity profile regardless of the targeted receptor within selective tissues. Hypophosphatemia, hypokalemia, hyponatremia, hypochloremia, and glucosuria were attributed to decreased reabsorption at the proximal tubule. Given the absence of glomerular lesions, proteinuria was also considered a likely consequence of decreased endocytic uptake in the proximal tubule.

In the first-in-man trial, all patients experienced increased serum creatinine and proteinuria, usually with casts, and these nephrotoxic changes precluded delivery of a full cycle in two out of six patients (33%). These changes were largely reversible without intervention beyond drug withdrawal, and no patient developed chronic renal dysfunction. No other clinical adverse events were observed.

Notably, we demonstrated selective BMTP-11 localization in bone marrow involvement in six out of six (100%) prostate cancer patients. This observation leaves no doubt that BMTP-11 reliably binds to prostate cancer following infusion and is consistent with its original identification in cancer patients.4-9 Furthermore, co-localization of tumor apoptosis and BMTP-11 in this setting confirms the pre-clinical activity of this ligand-directed drug candidate and other D(KLAKLAK)2-containing agents reported in animal models.12-17 One hopes that future studies with lead-optimized linkers, specific blockers, new formulations, and dose or schedule changes may mitigate reversible kidney toxicity resulting from renal proximal tubule uptake. As no lesions were observed in the glomeruli, we concluded toxicity was not due to renal clearance.

In summary, this limited phase zero trial with biology-driven endpoints, (i) demonstrates ligand-directed drug localization in human tumor samples--data that validate vascular targeting observations in pre-clinical models, (ii) narrows the dose-range and schedule for a formal phase I study, (iii) defines the acute toxicity profile, and (iv) suggests the possibility for clinical activity in prostate cancer patients with osteoblastic bone metastases. Despite our very small patient cohort in a small first-in-human study, anatomic localization has been clearly demonstrated, an upper limit on BMTP-11 dose has been established for this initially used “weekly x 4” schedule, and hints of drug efficacy have been observed. The present results provide justification for consideration of BMTP-11 as a targeted prototype drug against human prostate cancer and for the exploration of lower doses and/or alternative schedules to evaluate whether there might be a threshold below which the renal toxicity is minimized or abrogated. From a wider perspective, the translation from human-based discovery to first-in-human clinical trial provides an integrated paradigm for streamlined targeted drug development in human cancer.

Supplementary Material

Supp Material

Acknowledgments

FUNDING SUPPORT

This work was supported by the Gillson-Longenbaugh Foundation, the Marcus Foundation, the Prostate Cancer Foundation, and the National Institutes of Health (CA140388). We thank Erin Horne, Bih-Fang Pan, and Connie Sun for technical assistance.

Footnotes

AUTHORS’ CONTRIBUTIONS

Conception and design: R. Pasqualini, R.E. Millikan, D.R. Christianson, W.H.P. Driesen, R.J. Giordano, A. Hajitou, K.F. Barnhart, D.H. Hawke, E. Efstathiou, P.Troncoso, C.J. Logothetis, W. Arap.

Acquisition of data (e.g., provided animals, acquired and managed patients, provided facilities and/or materials): R. Pasqualini, R.E. Millikan, D.R. Christianson, W.H.P. Driessen, R.J. Giordano, A. Hajitou, K.F. Barnhart, V.D. Marcott, D.H. Hawke, N.M. Navone, E. Efstathiou, P.Troncoso, C.J. Logothetis, W. Arap.

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Pasqualini, R.E. Millikan, D.R. Christianson, M. Cardó-Vila, W.H.P. Driessen, R.J. Giordano, A. Hajitou, A.G. Hoang, S. Wen, K.F. Barnhart, W.B. Baze, D.H. Hawke, K-A. Do, N.M. Navone, E. Efstathiou, P. Troncoso, R.R. Lobb, C.J. Logothetis, W. Arap.

Writing, review, and/or revision of the manuscript: R. Pasqualini, R.E. Millikan, D.R. Christianson, M. Cardó-Vila, W.H.P. Driessen, R.J. Giordano, S. Wen, K.F. Barnhart, D.H. Hawke, E. Efstathiou, P. Troncoso, R.R. Lobb, C.J. Logothetis, W. Arap. All authors approved the final manuscript.

CONFLICT OF INTEREST DISCLOSURES

At the time of the study, the University of Texas M. D. Anderson Cancer Center (MDACC) and R.P., W.A. and R.R.L. owned equity stock in Alvos Therapeutics (Arrowhead Research Corporation; Pasadena, CA). The other authors declare that they have no competing interests.

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