Safety Profile After Prolonged C3 Inhibition

1.1. C3 inhibitor Cp40

Cp40 (dTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH₂, 1.8 kDa) was produced by solid-phase peptide synthesis as previously described [24]. Peptides were purified by reversed-phase high-performance liquid chromatography, characterized by mass spectrometry [24], and tested for the presence of endotoxin (< 0.03 EU/mL).

1.2. Injection of Cp40 into cynomolgus monkeys

A total of 33 cynomolgus monkeys (Macaca fascicularis) housed at the Simian Conservation Breeding and Research Center (SICONBREC, Makati City, Philippines) were used in this study. Monkeys (25 males and 8 females) were ~5 years of age, weighed ~4 kg, and were considered healthy after evaluation by an experienced veterinarian. Prior to the study, the animals were acclimatized for 2 weeks in the experimental room under conditions of controlled temperature (26 ± 4 °C), relative humidity (60 ± 25 %), and ventilation and a natural day and night cycle. Animals were provided with 100 g of food (standard monkey grower pellets, Jetstar Milling Corporation, Lipa Batangas, Philippines) daily and with water ad libitum (via water bottle) throughout the acclimation and observation periods. A fresh banana was given daily as supplementary diet. On the day of dosing, food was provided after the administration of the compound. Adult animals were injected subcutaneously or intravenously every 24 or 48 h with 2, 3, 4, or 20 mg/kg of Cp40 dissolved in sterile saline or water for injection containing 5% dextrose. Blood samples (1–2 ml) were collected from the femoral vein into EDTA-vacutainer tubes immediately before and at various time points after compound injection. Samples were centrifuged at 800 × g for 10 min to obtain plasma and immediately frozen at ≤ −80 °C until further analysis.

Full hematologic and biochemical evaluations, using a Mythic 18 VET- hematology analyzer and Microlab 300 semi-automated clinical chemistry analyzer, were also conducted on blood and serum samples collected from all animals in the study. The parameters measured were: white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, platelet count, thrombocrit, mean platelet volume, platelet distribution width, lymphocyte count, monocyte count, granulocyte count, prothrombin time, activated partial thromboplastin time, reticulocyte count, glucose, total cholesterol, triglycerides, blood urea nitrogen, creatinine, total protein, albumin, globulin, total bilirubin, amylase, glutamate oxaloacetate transaminase, glutamate pyruvic transaminase, alkaline phosphatase, gamma glutamyl transferase, magnesium, calcium, inorganic phosphorous, chloride, potassium, and sodium. The values obtained were compared with normal reference values previously obtained in cynomolgus monkeys [25].

Animals were sedated using ketamine (5–10 mg/kg) and xylazine (1–2 mg/kg) before blood collection. Atropine sulphate (0.04 mg/kg) was given prior to anesthesia. Where indicated, animals were euthanized, and full necropsy with histologic assessment was performed. All cynomolgus monkeys studies were performed in accordance with animal welfare laws and regulations, as approved by the Institutional Animal Care and Use Committee (IACUC). SICONBREC is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

1.3. Quantitation of Cp40 and C3 in the plasma

Levels of Cp40 in the plasma of cynomolgus monkeys were determined after methanol precipitation of Cp40 from plasma samples and quantification by reversed-phase liquid chromatography coupled with mass spectrometry, with a detection limit of 0.18 μg/ml (100 nM) [22]. The plasma concentration of C3 was determined using nephelometry as previously described [26].

1.4. Wound healing model

A model of wound healing was established in cynomolgus monkeys. Animals were anesthetized by an intramuscular injection of ketamine (10–12 mg/kg) plus xylazine (2 mg/kg) on the day of surgery. The skin on the back was cleaned, shaved, and sterilized with betadine followed by 4% chlorhexidine and 70% isopropyl alcohol. Four excisional punch biopsies (8- mm diameter and full skin thickness) were taken from the back of each animal. The borders of the wounds were marked using indelible, nontoxic ink and each site was covered with a sterile gauze dressing with Chlorhexidine (0.40% to 0.60%) for 2 h after surgery. The animals were then placed in a jacket (Lomir Biomedical, Inc., Quebec, Canada) that does not restrict mobility but prevents them from scratching the biopsy site and housed individually to prevent any potential wound disturbance by other animals. Ketorolac (0.5 mg/kg) was given twice a day to prevent pain.

Animals in the treatment group received the compstatin analog Cp40 (2 mg/kg subcutaneously, every 12 h for 7 days; n=4), and control animals were injected with vehicle (water plus 5% dextrose, same injection scheme; n=4). The size of each biopsy site was monitored daily until complete healing occurred and digitally documented. ImageJ2 software (National Institutes of Health) was used to calculate changes in wound area over time. Repair of the wound area was calculated as the percentage of area of the original wound.

Additional skin biopsies (12 mm) were collected at days 5 and 12 from the healing wounds; tissues were snap-frozen in liquid N₂ and stored at ≤ −80 °C until further use. These sections of skin biopsies were fixed in 10% formalin for hematoxylin and eosin (H&E) staining. The fixed tissues were dehydrated in graded concentrations of alcohol, cleared in xylene, and infiltrated and embedded in paraffin. Paraffin blocks for each animal were then sectioned at 5-µm thickness and stained with H&E for gross histologic examination. H&E slides were scanned with a D.SIGHT 200 fluo slide scanner (Menarini Diagnostic, Italy), and images of the sections were acquired at 2× magnification.

1.5. Measurement of residual complement activity in the plasma

The residual complement activity was assessed in plasma samples of cynomolgus monkeys from both Cp40-treatment and control groups using a standard hemolytic assay. Briefly, rabbit blood cells (5% v/v) were incubated with 10% plasma in GVB-Mg²⁺ buffer (150 μl total volume) for 1 h at 37 °C in a 96-well plate. The plate was spun down and 100 μl of supernatant was transferred to clean wells. Absorbance was measured at 405 nm and converted into % of activation, where 100% activation equals to the O.D. obtained in plasma samples collected at time point 0 h, previous to treatment initiation. The relative residual complement activity measured in plasma samples containing Cp40 was plotted against the peptide concentration using GraphPad Prism 5 (La Jolla, CA).

1.6. Quantitative Real-Time RT-PCR

RNA was extracted from skin punch biopsies or tissue specimens collected during necropsy of cynomolgus monkeys by using the RNeasy® Fibrous Tissue Mini (Qiagen, Germantown, MD) or RNeasy® Mini (Qiagen) kits, respectively. RNA concentration and purity were assessed using a NanoDropTM 2000c spectrophotometer (Thermo Fisher Scientific). Samples with an A₂₆₀/A₂₈₀ ratio of ~2.0 and A₂₆₀/A₂₃₀ ratio ≥1.8 were reverse-transcribed by using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). The resulting cDNA was further amplified using TaqMan® Fast Universal PCR Master Mix (Applied Biosystems) on a StepOnePlus Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. Custom TaqMan® Array 96 - Well FAST Plates (Applied Biosystems) were designed containing the following Macaca fascicularis-specific probes: Mf02789775_m1 (IL1B), Mf02789780_m1 (IL2), Mf00541746_m1 (IL4I1), Mf02789322_m1 (IL6), Mf02789706_g1 (IL8), Mf02789325_m1 (IL10), Mf02789753_m1 (IL12B), Mf03043053_m1 (IL13), Mf04367377_m1 (IL17A), Mf02877343_m1 (IL22), Mf02794722_m1 (C3), Mf04385270_g1 (CXCL5), Mf02787883_m1 (FGF2), Mf02788577_m1 (IFNG), Mf00966524_m1 (PDGFB), Mf04389740_m1 (SELE), Mf04384675_m1 (SELP), Mf00998133_m1 (TGFB1), Mf02789784_g1 (TNF), Mf04383655_g1 (VEGFB). Expression of target mRNA was normalized using the signal obtained from the amplification with the 18S probe (Hs99999901_s1).

1.7. Cp40 iodination- Rhesus monkey imaging study

In order to study the biodistribution of Cp40 in PET imaging studies, Cp40 was labeled with the [¹²⁴I] tracer. [¹²⁴I]KI (IBA; 3.75 mCi) in 20 mM NaOH was buffered with 100 mM HCl and 1 M HEPES, pH 7. The following were then added: carrier KI (VWR; 0.67 µmol), Cp40 (0.67 µmol) in 0.3% Tween-20, and Chloramine-T (0.67 µmol) in a total volume of 563 μl. Cp40 precipitated immediately when the Chloramine-T was added but returned to solution with the addition of 10% Tween-20 (70 µl) to the reaction. The reaction was allowed to proceed for 3.5 min at room temperature and then quenched by adding 30 mM sodium metabisulfite (1.0 µmol). The quenched reaction was immediately diluted in water (15 ml) and loaded onto a Sep Pak C18 light SPE cartridge. The cartridge was washed in water (3 ml), then peptide (1.9 mCi) was eluted in ethanol (1.5 ml). HPLC evaluation of the SPE eluent indicated recovery of 43% of the total radioactivity as [¹²⁴I]Cp40, i.e., 0.82 mCi; thus, the radiochemical yield was 0.82 mCi/3.75 mCi = 22%.

The SPE eluent was then reduced in volume to near dryness under a gentle stream of helium at room temperature. The dose was formulated without further purification of [¹²⁴I]Cp40. Cold Cp40 was added to achieve the desired formulation of 1.0 mCi [¹²⁴I] / 5.0 mg total Cp40/ 1.4 ml for the 2.55 kg animal, plus excess for transfer loss; the dose was syringe end-filtered (0.22 μm) into a sterile vial. HPLC evaluation of the final dose indicated 45% of total radioactivity as [¹²⁴I]Cp40.

1.8. Injection of [¹²⁴I]Cp40 and PET imaging

A female juvenile rhesus monkey, ~1.5 years of age (2.55 kg), was sedated with telazol (intramuscular; 5–8 mg/kg) and injected subcutaneously with 41.85 MBq (1.131 mCi) [¹²⁴I]Cp40 (0.20 mCi/mg specific activity; 5.66 Cp40 mass [mg]; ~1 ml). Blood samples (~2 ml, peripheral vessel) were collected into EDTA at −1 min, then at 0, +5, and +30 min and at 1, 4, 8, 24, 48, 72, and 240 h post-injection and counted to enable calculation of the percentage of the injected dose (%ID) per mL of plasma. PET/CT imaging (GE Discovery® PET/CT) was performed post-injection under telazol sedation: after 30 min (20-min duration of scan), after 4 h (20-min duration of scan) and after 1, 2, 3, and 10 days (40-min duration scan on each day). The animal was placed on the scan bed according to standard operating procedures and maintained in a radioactive monitoring housing area to address radioactive waste for the duration of the study.

Representative fused PET/CT images projections (MIP) from the scans acquired 1, 3, and 10 days after injection of [₁₂₄I]Cp40 were captured using MIM version 6.7 (MIM Software Inc., Cleveland, OH). The total activities residing in regions of high radiotracer concentration including the injection site, thyroids, gallbladder, liver, intestines, spleen, heart, and urinary bladder for each time point were determined from volumes of interest measurements of the PET images using Pmod v3.7 image analysis software package (PMOD Technologies Ltd., Zurich, Switzerland). The resulting regional total activities were decay-corrected and divided by the injected dose to yield percentages of injected doses for each time point. Regional %IDs were subsequently plotted as functions of time using GraphPad Prism 7 (La Jolla, CA).

This study was conducted at the UC Davis California National Primate Research Center. All animal procedures conformed to the requirements of the Animal Welfare Act and protocols were approved prior to implementation by the UC Davis IACUC. UC Davis is an AAALAC-accredited institution.

2.1. In vivo distribution of Cp40 is associated with the presence of C3

The pharmacokinetic profile of the C3 inhibitor Cp40 has been extensively investigated and shows a plasma half-life of ~40 h after a single subcutaneous dose of 2 mg/kg. The peptide reaches maximum concentration in the plasma at 2 h post-injection, followed by a slow elimination phase and resulting in complete C3 saturation for ~4.5 h [22, 24]. To further understand the biodistribution of Cp40, a rhesus monkey was dosed subcutaneously with 42 MBq of [¹²⁴I]-labeled Cp40 (2.2 mg/kg of Cp40), followed by PET/CT scans performed over time to measure changing radioactivity concentrations in the organs. Fused PET/CT images of the in vivo distribution of [¹²⁴I]Cp40 are displayed in Fig. 1A, B and percentages of injected doses in the regions with the highest concentrations of [¹²⁴I]-labeled Cp40 are shown as a function of time (Fig. 1C). The highest percentage of the injected dose was located at the subcutaneous injection site (Fig. 1A), indicating the presence of a depot compartment exhibiting a delayed release of radiotracer into the circulation, with percentages of injected dose remaining at the injection site steadily decreasing from 64 %ID after 30 minutes to 19 %ID after 10 days. Highest regional uptakes of [¹²⁴I]Cp40 based on the areas under the curve (AUC) for percentages of the injected dose over time in descending order were; the injection site, thyroid, gallbladder, liver, intestines, spleen, heart, and urinary bladder (7683, 152, 110, 87, 46, 31, 21, and 11 %•hr) (Fig. 1C). The fourth highest regional amount of [¹²⁴I]Cp40 by AUC was detected in the liver (Fig. 1C), the main source of C3 in the body [27]. High levels of [¹²⁴I]Cp40 in the gallbladder and intestines may be due to hepatobiliary excretion of the PET radiotracer (Fig. 1). The radioactivity in the heart may primarily reflect the presence of [¹²⁴I]Cp40 in the blood since the highest uptakes in the heart of 0.34 %ID and in blood plasma of 0.05%ID/mL both peaked at 4 hours post-injection (Fig. 1). Ten days after injection of [¹²⁴I]Cp40, radiotracer uptake was primarily observed in the injection site, thyroids, and gallbladder (Fig. 1). In general, radiotracer concentrations decreased over time everywhere except for the thyroid, which exhibited increasing concentrations that may be due to the iodine radiotracer attached to Cp40 [28] (Fig. 1). Nasal accumulation of [¹²⁴I]Cp40 was faintly visible in the sagittal PET/CT image acquired 1 day post-injection (Fig. 1A) and is a typical finding for compounds labeled with iodine [29]. Overall these data, with the exception of high thyroid uptake of [¹²⁴I]Cp40 likely due to the iodine radiolabel, indicate that the body distribution of Cp40 is associated with the presence of C3, concentrating in organs that accumulate blood (heart, spleen, liver) and produce C3 (liver, spleen).

2.2. In vivo C3 inhibition during a one-week period

We next investigated whether systemic blockage of C3 for a period of 7 days would affect immune and biochemical parameters measured in the blood of cynomolgus monkeys. To this end, monkeys were dosed subcutaneously with 2 mg/kg of Cp40 every 12 h for a total of 7 days; control animals were injected with vehicle (water plus 5% dextrose) according to the same schedule (Fig. 2A). During the 7-day period, the plasma concentrations of C3 remained constant in both the control and Cp40-treatment groups (Fig. 2B). Also, the Cp40 dosing schedule ensured constant levels of peptide that exceeded the concentration of C3, indicating complete saturation of plasma C3 by the Cp40 inhibitor (Fig. 2B, right panel). Complete inhibition of C3 over the 7-day period was confirmed by the absence of residual complement activity in the plasma of the animals treated with Cp40, whereas plasma samples from control animals retained full ability to activate complement (Fig. 2C).

Given that systemic inhibition of C3 was achieved for a period of 1 week, we then profiled immunologic, coagulation and biochemical parameters in the blood of monkeys at day 7 post-treatment and compared with the baseline values obtained prior to Cp40 injection (day 0) (Fig. 2D). Despite the C3 inhibition, no differences in hematological, coagulation and biochemical parameters were identified in the blood of animals treated with Cp40 when compared with those injected with vehicle alone (data not shown). Furthermore, blood cell counts, including counts of total white blood cells, red blood cells, platelets, and monocytes, were within the normal range and were not influenced by the Cp40 treatment (Fig. 2D). Although both control and treatment groups showed an elevated granulocyte count and decreased lymphocyte count at day 7 when compared with baseline values, the values remained within the normal range observed in healthy animals (Fig. 2D).

In parallel, at 3 h after the initial injection of Cp40, the local immune response was triggered in these animals by skin biopsies. A total of four punch biopsies were taken from the dorsal side of each animal, and the size of each site was monitored daily until complete healing had occurred (Fig. 3A). Notably, the wounds showed no signs of infection (no antibiotics administered). In line with these observations, mRNA expression levels of cytokines (IL-2, IL-6, IL-8, IL-10, IL-13, IL-17, IL-22, CXCL5, TNF, TGF-β) and other molecules associated with wound healing (SELP, PDGFB, FGF2, SELE, LEGFB) were similar in skin biopsies from the control and treated animals, indicating an absence of local active infection despite Cp40 treatment (data not shown). Gross histologic observation showed no appreciable differences between the wounds biopsied from control and Cp40-treated animals (Fig. 3B). Skin biopsies showed sites of necrosis and ulceration, vascular and fibroblastic proliferation, and infiltration of inflammatory cells in the dermis at day 5 post-biopsy and an inflammatory exudate, scarring of the dermis, and reconstruction of the epidermis at day 12 post-biopsy (Fig. 3B), illustrating the classic inflammatory and proliferative phases of wound healing [30]. Further, C3 inhibition did not result in delayed healing. Instead, Cp40 treatment was associated with a trend toward faster wound healing than in control animals (Fig. 3C), suggesting that the therapeutic inhibition of C3 may modulate the local wound environment and promote the healing process.

2.3. In vivo C3 inhibition during a 2-week period

Cp40 treatment and consequent C3 inhibition was then extended for 2 weeks. During this extended treatment, systemic toxicity was also evaluated in response to the administration of a dose 10-fold higher than the established therapeutic dose (2–4 mg/kg). Cynomolgus monkeys were therefore dosed daily with intravenous infusions of 20 mg/kg of Cp40 for a total of 14 days, whereas control animals were injected with vehicle alone (Fig. 4A). The Cp40 dosing schedule resulted in a discrete accumulation of peptide in the circulation (from ~6 μM at day 0 to ~8 μM at day 12) and ensured the saturation of C3 in the plasma over the course of the entire treatment (Fig. 4B). After the last injection, on day 14, levels of plasma Cp40 rapidly decreased and were completely cleared by day 24 (Fig. 4B).

The animals were observed for a total of 40 days, and no mortality, clinical signs, effects on body weight and food consumption, or changes in clinical parameters such as body temperature, heart and respiratory rate, urine output, or blood pressure were observed in the animals treated with Cp40 (data not shown), indicating that the compound is well tolerated and does not induce systemic toxicity following repeated intravenous administration. Similarly, hematological (Fig. 4C), coagulation and blood chemistry (data not shown) clinical tests did not detect any changes directly induced by the peptide. Upon scheduled endpoint, histopathologic examination of the lung showed isolated areas of hemosiderosis (4 of 6 animals), atelectasis (1 of 6 animals) and anthracosis (2 of 6 animals). Signs of hemosiderosis and moderate to severe pulmonary edema were also found in animals from the control group that had been treated with vehicle alone, indicating that these observations are unrelated to the treatment (Supplementary Table 1). With the exception of the lungs, all other organs were unremarkable with no adverse finding (Supplementary Table 1).

Expression levels of C3 mRNA in the spleen and liver of animals treated with Cp40 were similar to the levels found in tissues from those in the control group (Figure 4D, left panel). Similarly, no changes were observed in the mRNA expression of the proinflammatory cytokines IL-1β, IL-6, IL-10, IL-12, or IFN-γ in the spleen of Cp40-treated animals when compared with control spleens (Figure 4D, right panel), confirming that none of the treated animals showed any signs of infection or autoimmune disease during the trial period or during the 28 days following the administration of the peptide. Interestingly, an increase in the mRNA expression levels of the IL-22 cytokine was observed in the spleens of the Cp40-treated animals when compared with the expression of the same cytokine in the control group (Figure 4D, right panel).

2.3. In vivo C3 inhibition during a 3-month period

Next, systemic toxicity and treatment-associated effects were evaluated during a 3-month period in which cynomolgus monkeys were injected daily with 3 mg/kg of Cp40 subcutaneously (Fig. 5A); animals in the control group were injected with vehicle alone. Again, saturation of C3 concentrations in the plasma was observed during the entire treatment period (Fig. 5B).

No mortality, clinical signs, effects on body weight and food consumption, or changes in clinical parameters such as body temperature, heart and respiratory rate, urine output, or blood pressure were observed in the animals treated with Cp40 (data not shown), indicating that the compound is well tolerated and does not induce systemic toxicity following long-term administration. Similarly, hematological (Fig. 5C), coagulation and blood chemistry (data not shown) clinical tests did not detect any changes directly induced by the peptide. Upon necropsy, histopathologic examination of the subcutaneous tissue showed local inflammatory infiltrate in 6 of 8 animals in the treatment group (Supplementary Table 2), likely a consequence of the repeated subcutaneous injections with peptide. Animals from both the treatment and control groups showed lung atelectasis (Supplementary Table 2), a consequence of lung collapse during opening of the chest cavity. Apart from the subcutaneous tissue, no other organ indicated any adverse findings (Supplementary Table 2).