Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

CRP¹ is a major component of the acute-phase response. As part of the immediate defense system of the body, CRP serum concentrations can increase up to 1000-fold within 24-72 hr of an infection or tissue insult (Kushner, 1982; Pepys and Baltz, 1983). The in vitro biological activities of CRP include complement activation (Siegel et al., 1974; Kaplan and Volanakis, 1974), opsonization (Nakayama et al., 1982; Kilpatrick and Volanakis, 1991), and activation of macrophages for tumoricidal activity (Zahedi and Mortensen, 1986; Barna et al., 1987, 1988). Both in vitro and in vivo, CRP has been shown to bind to and affect the clearance of nuclear antigens (DuClos, 1996). However, no definitive in vivo function has been identified for CRP.

Our laboratory has established that CRP can exist in a conformationally and antigenically distinct form that we call mCRP (Potempa et al., 1983, 1987). mCRP has been shown to be a naturally occurring protein in various tissues throughout the body (Rees et al., 1988; Egenhofer et al., 1993; Radosevich et al., 1996). mCRP has immunostimulatory activities that are different from those reported for the native pentameric CRP. These include modulation of leukocyte, monocyte, and platelet activities in vitro (Potempa et al., 1988), potentiation of megakaryocyte growth in vitro, and stimulation of thrombopoietic activity in vivo (Potempa et al., 1996).

One of the major physical differences between CRP and mCRP is the difference in their solubility characteristics. Whereas CRP is soluble in buffers of physiological ionic strength and pH, mCRP is maximally soluble in solutions of low ionic strength and more alkaline pH. When added to solutions of physiological pH and ionic strength, mCRP forms an opalescent suspension. Our laboratory has studied both mCRP-sol and mCRP-susp forms as biological response modifiers in various animal disease models. In addition to assessment of the in vivo efficacy of mCRP as a potential therapeutic agent, it is necessary to determine in vivo distribution characteristics and clearance mechanisms.

The in vivo plasma clearance of ¹²⁵I-CRP was previously reported. The plasma half-life of ¹²⁵I-CRP was ~4 hr in mice and rats (Baltz et al., 1985) and ~7 hr in rabbits (Challadurai et al., 1983; Rowe et al., 1984). Plasma and whole-body turnover of human ¹²⁵I-CRP has also been reported for normal and diseased volunteers (Vigushin et al., 1993). The clearance closely approximated a monoexponential function, with ~90% of injected radioactivity being recovered in urine after 7 days. The half-life was 19 hr in normal volunteers and was unchanged in patients with rheumatoid arthritis, systemic lupus erythematosis, bacterial infections, or malignant neoplasia. Furthermore, scintographic analyses using ¹²³I-CRP in 10 patients with prominent inflammation and tissue damage revealed no selective localization of labeled CRP to any tissue or organ; the distribution of label was confined to the blood pool.

This is the first report of the PK/BD characteristics of mCRP. We used ¹²⁵I-labeled CRP to prepare both mCRP-sol and mCRP-susp for injection into normal male mice. The PK parameters and tissue BD for both forms of mCRP were then compared with those of the more widely studied CRP molecule.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Preparation of Labeled Proteins. CRP was isolated and purified from human ascitic fluids by calcium-dependent phosphorylcholine affinity chromatography, using phosphorylcholine-substituted Bio-Gel resin (Bio-Rad Laboratories, Richmond, CA), and ion exchange chromatography, according to previously published procedures (Potempa et al., 1987). Purified CRP at 1 mg/ml was dialyzed into 25 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl and 2 mM CaCl₂. CRP was iodinated using Iodo-Bead technology (Pierce Chemical Co., Rockford, IL), as follows. For every 1 ml of CRP solution, six beads were washed with iodination buffer (25 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl and 5 mM CaCl₂,). Na¹²⁵I was diluted to 3 mCi/ml of iodination buffer and added to the beads. After 5 min, 1 ml of CRP (at 1 mg/ml) was added and allowed to incubate for 5 min at room temperature. The iodinated protein was removed and passed through a Sephadex G-75 column (Pharmacia, Piscataway, NJ), to separate free label from protein-bound label and to equilibrate the ¹²⁵I-CRP solution in 10 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl and 2 mM CaCl₂.

Animals. Male CD-1 mice were purchased from Charles River Laboratories, Canada. The mice (age, 35-49 days; weight, 26-31 g) were quarantined for approximately 2 weeks and then examined for signs of disease or injury before the start of the study. Mice were randomized into six groups. Each mouse received a single bolus injection (volume, 0.14-0.31 ml), via the tail vein, of a mixture of unlabeled and labeled CRP, mCRP-sol, or mCRP-susp. Dose volumes were calculated based on the pretreatment body weight and were rounded to the nearest 0.01 ml. For each form of CRP studied, one group of mice had blood samples taken at 5 min, 2 hr, and 4 hr, urine and feces collected over 0-4 hr, and tissue samples obtained, after sacrifice, at 4 hr. Another group of mice had blood samples taken at 30 min, 8 hr, and 24 hr, urine and feces samples collected over 0-24 hr, and tissue samples obtained at 24 hr.

Data Analysis. PK parameters were calculated for each form of CRP by noncompartmental analysis using statistical moment theory (Riegelman and Collier, 1980). The AUC and AUMC were calculated by the linear trapezoidal rule, using the following equations. Because of the nature of the plasma data, the elimination rate constant (K_e) could only be estimated as the slope of the line formed by the last two blood collection time points. The estimated MRT was calculated from the AUMC/AUC ratio. The estimated half-life of elimination was calculated as 0.693/K_e. The estimated CL_T was calculated as the mean dose/AUC ratio. The estimated V_ss was calculated as CL_T × MRT.

Tissue Distribution Analysis. The tissue and organ accumulation of radiolabeled protein in 4 and 24 hr after iv administration was estimated by assuming that the ¹²⁵I-labeled protein complex was stable. The distribution was expressed either as the percentage of the injected dose recovered in each collected tissue or organ or as the percentage of the injected dose recovered in each tissue or organ divided by the weight of that tissue or organ (in grams). The values therefore have units of percent or percent per gram, respectively. For plasma, urine, or feces, the volume (in milliliters) was used instead of the weight, i.e. values of percent per milliliter are reported instead of percent per gram.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

PK. The PK clearance of ¹²⁵I-CRP was biexponential (fig. 1), with a rapid early phase and a slower later phase. The half-life of ¹²⁵I-CRP was calculated as 4.7 hr. At the first time point measured, i.e. 5 min, ~50% of injected ¹²⁵I-CRP was recovered in plasma. In contrast, the clearance of both ¹²⁵I-mCRP-sol and ¹²⁵I-mCRP-susp was very rapid; at the 5-min time point, <5% of the injected label could be recovered in plasma (fig. 1, inset).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Comparison of the plasma concentration-time curves for 125 I-mCRP, 125I-mCRP-sol, and 125I-mCRP-susp. CD-1 mice were injected with 0.5-1.5 × 106 cpm of 125I activity in a 2.5 mg/kg dose consisting of a mixture of labeled and unlabeled protein. Plasma aliquots obtained at 5 min, 30 min, 2 hr, 4 hr, 8 hr, and 24 hr were analyzed for 125I activity. The mean values from three mice in each group were plotted. Inset, percentage of injected label recovered in plasma.

BD. Figs. 2 and 3 show the tissue distribution of ¹²⁵I-CRP and ¹²⁵I-mCRP-sol, at 4 and 24 hr after iv injection into mice, expressed either as the percentage of the injected dose or as the percentage of the injected dose per gram for each tissue (or organ). At 4 hr, the greatest amount of recovered ¹²⁵I-CRP was found in plasma (12% of injected dose). Skeletal muscle (7%), liver (6%), urine (6%), skin (5%), and femur bone (5%) were the other predominant sites for the uptake of ¹²⁵I-CRP (fig. 2A). The greatest concentrations of label (expressed as percentages of the injected dose per gram) were found in the urine and plasma (39%/ml and 14%/g, respectively) (fig. 3A). At 24 hr, the percentage of ¹²⁵I-CRP in urine was significantly increased (fig. 2A). The rank order of accumulation was urine (22%) > bone marrow (8%) > femur bone (6%) > skeletal muscle (5%). There was a notable increase in the concentration of radiolabel localized to the bone marrow at 24 hr (27%/g), relative to the 4-hr time point (4.9%/g) (fig. 3A).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Tissue and organ accumulation of 125I-CRP at 4- and 24-hr time points, expressed as percentages of the injected dose (A) or percentages of the injected dose per gram (plasma, urine, and feces values are expressed as percentages of the injected dose per milliliter) (B). L.N., lymph nodes.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Tissue and organ accumulation of 125I-mCRP-sol at 4- and 24-hr time points, expressed as percentages of the injected dose (A) or percentages of the injected dose per gram (plasma, urine, and feces values are expressed as percentages of the injected dose per milliliter) (B). No urine sample was obtained for analysis at the 4-hr time point. L.N., lymph nodes.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The present study compares the clearance characteristics of ¹²⁵I-mCRP-sol and ¹²⁵I-mCRP-susp with those of ¹²⁵I-CRP after iv injection of each protein into normal CD-1 mice. CRP is widely known as the prototypical acute-phase reactant; its plasma levels are markedly increased within hours after acute, tissue-damaging, inflammatory events (Kushner, 1982; Pepys and Baltz, 1983). mCRP is a conformationally modified form of CRP that is naturally occurring, primarily as a tissue-based (rather than a serum-based) form of CRP (Egenhofer et al., 1993; Potempa et al., 1983, 1987; Rees et al., 1988; Radosevich et al., 1996).

The plasma clearance of ¹²⁵I-CRP measured here is biexponential, with a half-life of 4.7 hr (282 min); this value closely agrees with previous reported values for the CRP molecule (Baltz et al., 1985). The half-life for CRP injected into human subjects was reported to be 19 hr (Vigushin et al., 1993), and the same plasma half-life values were measured in normal control subjects and in patients documented to have infections, neoplasms, rheumatoid arthritis, or systemic lupus erythematosis. This result was surprising, because maladies such as those described are known to stimulate increased blood levels of CRP; it was thought that localized disease would produce increased binding sites for CRP, which would therefore be cleared more quickly.

In contrast to ¹²⁵I-CRP, ¹²⁵I-mCRP was cleared from the plasma very rapidly; >95% of the injected protein was removed from plasma at the 5-min time point. The short plasma half-life of the drug is not the result of rapid clearance from the body, because 80-100% of the radiolabel injected with mCRP could be accounted for in samples collected at the 24-hr time point. These data suggest that mCRP, in contrast to CRP, is immediately taken up into tissues within the first few passages through the circulation.

mCRP is a protein that is soluble in low-ionic strength solutions and forms self-aggregates when placed in buffers of physiological ionic strength (Potempa et al., 1983, 1987). Antigens cross-reactive with mCRP are found to be abundantly expressed at the intima, media, and adventitia of a number of normal blood vessels. Immunohistochemical reactivity is also found in the fibrous capsule of the adrenal glands and tonsils, in the fibrous trabeculae of the spleen, and in skin and skeletal muscle (Radosevich et al., 1996). These data are consistent with the view that mCRP is a natural component of the extracellular matrix of reticuloendothelial tissues. The fact that the majority of injected ¹²⁵I -mCRP is rapidly removed from the circulation may reflect a strong tendency for the mCRP conformer to partition into tissues where naturally occurring mCRP is found. This result is supported by the large V_ss values calculated for mCRP (23-27 ml) and by the BD results. Drugs that are sequestered by tissues exhibit large V_ss values (Benet and Sheiner, 1988). CRP, in contrast, is primarily a protein of the circulatory system, having a comparatively small V_ss (2.35 ml).

mCRP exhibits an ~10-fold larger V_ss than does CRP, and estimated CL_T and MRT values were calculated to be ~5- and ~2-3-fold greater, respectively, than those for CRP. These data, together with the finding that all radiolabel associated with mCRP could be accounted for at the 24-hr time point, indicate that, by accessing tissues, mCRP is cleared at a slower rate than CRP.

TCA precipitation of plasma samples showed that there was a generally uniform recovery of ~72-84% of ¹²⁵I-CRP from plasma through the 8-hr time point after treatment. However, TCA precipitation of ¹²⁵I-mCRP-sol or ¹²⁵I-mCRP-susp plasma samples obtained up to the 4-hr time point showed only 49 and 63%, respectively, of the label remaining protein bound (data not shown). The lower percentage of TCA-recoverable label in the <5% subfraction of injected ¹²⁵I-mCRP recovered in the plasma suggests that the label might have dissociated from the protein, that mCRP in plasma might have been degraded faster than mCRP partitioned into tissues, or that nonlinear protein binding might have occurred in vivo. These data suggest that the ¹²⁵I-mCRP recovered in plasma at 4 hr might be a distinctive subpopulation of ¹²⁵I-mCRP or a metabolic breakdown product of the prepared reagent.

Tissue distribution of ¹²⁵I-CRP, ¹²⁵I-mCRP-sol, and ¹²⁵I-mCRP-susp was generally unremarkable, except for the interesting selective BD of both forms of mCRP to the bone marrow. The early distribution measured at 4 hr after iv injection of all forms of ¹²⁵I-(m)CRP tested was found in the skeletal muscle, liver, and skin. At 24 hr, whereas 7% (equivalent to 27%/g) of ¹²⁵I-CRP was distributed to the bone marrow, ~25% (equivalent to 92%/g) of both ¹²⁵I-mCRP-sol and ¹²⁵I-mCRP-susp was accumulated in the bone marrow. Thus, the BD results corroborate the PK estimates that mCRP, to a greater extent than CRP, is rapidly cleared from the blood compartment and enters tissue, especially bone marrow, where a large portion of it remains for at least 24 hr.

It is noteworthy that a key component of the bone marrow is the stroma, which is composed, in part, of reticular proteinaceous fibers that are similar to those found in fibrous tissues of the systemic reticuloendothelial system (Allen et al., 1990; Mohammad and Asai, 1993). The physical characteristics of mCRP as a protein that can self-associate into a matrix-like protein (Motie et al., 1996) may relate to its physiological role as a component of the extracellular matrix of tissues.

The finding that mCRP accumulates in the bone marrow for up to 24 hr after injection is noteworthy in light of our report that mCRP can stimulate megakaryocytopoiesis in mice (Potempa et al., 1996). Drugs that accumulate in a given tissue are believed to serve as a reservoir for prolonged drug action in that tissue or at another site (reached through the circulation) (Benet and Sheiner, 1988). The accumulation of radiolabeled mCRP in the bone marrow agrees with this organ being the site of increased hematopoietic activities and this drug having stimulatory effects on megakaryocytopoiesis. Furthermore, because mCRP can be formed from CRP under a variety of conditions (Potempa et al., 1987), the accumulation of ¹²⁵I-CRP in vivo in the bone marrow at 24 hr might indicate that some injected CRP was converted in situ to mCRP, which was then localized to this organ. The fact that there was increased accumulation of label in the bone marrow at 24 hr, although there was a very low level of radioactivity associated with mCRP in the circulation, may reflect capacity-limited clearance of mCRP. Because of its low aqueous solubility, injected mCRP may be sequestered (as a noncovalently associated aggregate) into tissues, where it serves as a reservoir for circulating protein. The tissue-associated mCRP would be in equilibrium with the plasma mCRP, which would slowly and continuously be sequestered into specific (e.g. bone marrow) tissues.

Hutchinson et al. (1994) reported the accumulation of ¹²⁵I-tyramine cellobiose-labeled CRP in hepatocytes of mice and rabbits 24 hr after iv injection. They suggested that the liver is the main organ for pentraxin catabolism. The same group reported that the kidney is the primary organ for excretion of CRP-associated radiolabel in humans (Vigushin et al., 1993). Our results indicate that the final excretory pathway for all forms of radiolabeled CRP appears to be through the urinary tract, presumably after filtration through the kidney. At the 24-hr time point, 40, 78, and 55%/ml of ¹²⁵I-CRP, ¹²⁵I-mCRP-sol, and ¹²⁵I-mCRP-susp, respectively, were found in the urine. We propose that the lack of substantial accumulation of radiolabel in the kidneys may be a sign of the low toxicity associated with the injection of mCRP in both soluble and suspended forms.

This study demonstrates that CRP and both forms of mCRP can be safely injected iv into mice, where they are cleared from the body and excreted in the urine. The selective localization of mCRP to tissues, especially the bone marrow, provides a clue to the bioactivity of this protein as a thrombopoietic factor (Potempa et al., 1996), as well as an amplifying factor in the leukocyte and platelet responses (Potempa et al., 1988). The therapeutic applications of mCRP in the treatment of various immunological diseases are under study.