Synthesis and biological evaluation of phosphonate analogues of 1α, 25-dihydroxyvitamin D3

A new series of phosphonate side chain analogues of 1alpha,25-dihydroxyvitamin D3 (1) have been synthesized. Antiproliferative activities of theses analogues (8a,b and 9a,b) using human keratinocyte cell shows that analogues which have natural A-ring show higher activity than unnatural A-ring series and almost equally active to 1alpha,25-Dihydroxyvitamin D3 (1) at 1 microM level.     

Cytochrome P450 3A-mediated microsomal biotransformation of 1α,25-dihydroxyvitamin D3 in mouse and human liver: drug-related induction and inhibition of catabolism

The biological activities of vitamin D(3) are exerted through the dihydroxy metabolite of vitamin D(3) [1α,25(OH)(2)D(3)]. Hepatic biotransformation of 1α,25(OH)(2)D(3) by cytochrome P450 (P450) enzymes could be an important determinant of bioavailability in serum and tissues. In the present study, we investigated the comparative biotransformation of 1α,25(OH)(2)D(3) in mouse and human liver microsomes and determined the effects of commonly used drugs on the catabolism of 1α,25(OH)(2)D(3). Severe symptoms of vitamin D deficiency have historically been observed in patients who received dexamethasone. To compare the effects of clinically important glucocorticoids with hepatic biotransformation of 1α,25(OH)(2)D(3), adult male CD-1 mice were given intraperitoneal injections of either vehicle (50% ethanol), dexamethasone (80 mg/kg per day), or prednisone (80 mg/kg per day) for three consecutive days. Hydroxy metabolite formation pattern and the extent of substrate depletion were similar in mouse liver microsomes (MLM) from vehicle- or prednisone-treated mice, whereas treatment with dexamethasone led to the emergence of additional metabolites and increased substrate depletion, as determined by liquid chromatography/mass spectrometry. The metabolite formation profile in vehicle-treated mice was different from that of human liver microsomes (HLM). Selective P450 chemical inhibitors have demonstrated that CYP3A isoforms are responsible for the microsomal biotransformation of 1α,25(OH)(2)D(3) in MLM. Coincubation of 1α,25(OH)(2)D(3) with commonly used drugs led to approximately 60 to 100% inhibition of CYP3A4-mediated catabolism of 1α,25-(OH)(2)D(3) in HLM. A species-based difference was identified between CYP3A-mediated hepatic microsomal metabolism of 1α,25(OH)(2)D(3) in humans and mice. We have shown that the clinical importance of glucocorticoids differentially modulates catabolism of active vitamin D(3) and that commonly used drugs could affect vitamin D homeostasis.     

An o-aminoanilide analogue of 1α,25-dihydroxyvitamin D(3) functions as a strong vitamin D receptor antagonist

Vitamin D receptor (VDR) antagonists have therapeutic potential in treatment of allergic conditions and hypercalcemia driven by granulomatous diseases. We have identified an o-aminoanilide analogue of the hormonal form of vitamin D with a dienyl side chain that functions as a strong VDR antagonist. Modeling studies indicate that antagonism arises from side chain rigidity, when compared to a more flexible saturated analogue, which interferes with H12 folding/alignment.     

Synthesis and biological evaluation of 1α,25-dihydroxyvitamin D(3) analogues hydroxymethylated at C-26

We designed by docking and synthesized two novel analogues of 1α,25-dihydroxyvitamin D(3) hydroxymethylated at C-26 (2 and 3). The syntheses were carried out by the convergent Wittig-Horner approach via epoxide 12a as a common key intermediate. The antiproliferative and transactivation potency of the compounds was evaluated in colon and breast cancer cell lines. The analogues showed a similar but reduced activity compared to 1,25(OH)(2)D(3). Analogue 3 was more potent than analogue 2, and in some assays it exhibited potency similar to that of the natural ligand.     

1α,25-dihydroxyvitamin D3 on intestinal transporter function: studies with the rat everted intestinal sac

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃) treatment (2.56 nmol/kg i.p. daily×4) increased PepT1, Mrp2, Mrp4, Asbt, but not Mdr1/P‐gp in the rat small intestine. In this study, the intestinal everted sac technique, together with various select probes: mannitol (paracellular transport), glycylsarcosine (PepT1), 5(and 6)‐carboxy‐2′,7′‐dichlorofluorescein (CDF) diacetate (precursor of CDF for Mrp2), adefovir dipivoxil (precursor of adefovir for Mrp4) and digoxin (P‐gp) was used to examine the functional changes of these transporters. After establishing identical permeabilities (P_app) of mannitol for the apical‐to‐basolateral (A‐to‐B) and basolateral‐to‐apical (B‐to‐A) directions at 20 min in 1,25(OH)₂D₃‐treated vs. vehicle‐treated duodenal, jejunal and ileal everted sacs, a significant enhancement of net A‐to‐B transport of glycylsarcosine in the duodenum, increased B‐to‐A transport of CDF and A‐to‐B and B‐to‐A transport of adefovir in the jejunum were observed with 1,25(OH)₂D₃ treatment. However, the A‐to‐B and B‐to‐A transport of digoxin in the ileum was unchanged. These changes in transporter function in the rat intestinal everted sac corresponded well to changes in proteins that were observed previously. This study confirms that the rat intestinal PepT1, Mrp2 and Mrp4, but not P‐gp are functionally induced by 1,25(OH)₂D₃ treatment via the vitamin D receptor (VDR). Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of 1α,25-dihydroxyvitamin D3 on transport and metabolism of adefovir dipivoxil and its metabolites in Caco-2 cells

Effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), natural ligand of the VDR, on the fates of adefovir dipivoxil (P-gp substrate) and its metabolites, mono(POM)-PMEA and adefovir (MRP4 substrate), were investigated in Caco-2 cells. After 1,25(OH)2D3-treatment, higher apical efflux of adefovir was observed after a 60 min incubation of adefovir divipoxil. Changes in these washout studies were predicted by a catenary model for the Caco-2 monolayer that described a higher MRP4 activity with 1,25(OH)2D3 treatment, as confirmed by Western blotting. Moreover, 1,25(OH)2D3 treatment (100 nM for 3 days) resulted in increased basolateral (B) to apical (A) (B-to-A) transport of adefovir dipivoxil but an unchanged A-to-B flux, rendering an elevated efflux ratio (EfR) (from 1.97 to 3.19). The EfR values in control and 1,25(OH)2D3-treated groups in these transport studies were reduced to 1.32 and 1.57, respectively, in the presence of verapamil (50 μM), the P-gp inhibitor. The B-to-A transport of the metabolite, adefovir, was increased in 1,25(OH)2D3-treated cells in the presence of verapamil, whereas the A-to-B and B-to-A transport of mono(POM)-PMEA remained unchanged. But the verapamil and 1,25(OH)2D3 treatments failed to alter rates of sequential metabolism of adefovir dipivoxil in cell lysate. The composite data established that 1,25(OH)2D3 treatment increased both P-gp and MRP4 transport activities without affecting the metabolism of adefovir dipivoxil by esterases. Moreover, an asymmetric appearance of metabolites, being higher with apical application, was observed. According to the catenary model, the asymmetry is suggestive that esterases are predominantly localized on the apical membrane and within the cell.     

PKPD modelling to predict altered disposition of 1α,25-dihydroxyvitamin D3 in mice due to dose-dependent regulation of CYP27B1 on synthesis and CYP24A1 on degradation

Background and Purpose

Concentrations of 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active ligand of the vitamin D receptor, are tightly regulated by CYP27B1 for synthesis and CYP24A1 for degradation. However, the dose-dependent pharmacokinetic (PK)-pharmacodynamic (PD) relationship between these enzymes and 1,25(OH)₂D₃ concentrations has not been characterized.

Experimental Approach

The pharmacokinetics of 1,25(OH)₂D₃ were evaluated after administration of single (2, 60 and 120 pmol) and repeated (2 and 120 pmol q2d ×3) i.v. doses to male C57BL/6 mice. mRNA expression of CYP27B1 and CYP24A1 was examined by quantitative PCR and 1,25(OH)₂D₃ concentrations were determined by enzyme immunoassay.

Key Results

CYP27B1 and CYP24A1 changes were absent for the 2 pmol dose and biexponential decay profiles showed progressively shorter terminal half-lives with increasing doses. Fitting with a two-compartment model revealed decreasing net synthesis rates and increasing total clearances with dose, consistent with a dose-dependent down-regulation of renal CYP27B1 and the induction of renal/intestinal CYP24A1 mRNA expression. Upon incorporation of PD parameters for inhibition of CYP27B1 and induction of CYP24A1 to the simple two-compartment model, fitting was significantly improved. Moreover, fitted estimates for the 2 pmol dose, together with the PD parameters as modifiers, were able to predict profiles reasonably well for the higher (60 and 120 pmol) doses. Lastly, an indirect response model, which considered the synthesis and degradation of enzymes, adequately described the PK and PD profiles.

Conclusions and Implications

The unique PK of exogenously administered 1,25(OH)₂D₃ led to changes in PD of CYP27B1 and CYP24A1, which hastened the clearance of 1,25(OH)₂D₃.Tables of Links

Cellular and paracellular calcium transport in the rat ileum and the influence of 1α,25-dihydroxyvitamin D3 and dexamethasone

Summary Concentration dependence of unidirectional calcium fluxes across the rat ileum freed from the serosa and the muscularis externa were measured in a modified Ussingchamber. Mucosa (m) to serosa (s) calcium flux showed a saturable component, whereas s to m calcium flux was linearly related to the calcium concentration between 0.125 mmol/1 and 5 mmol/1. At all calcium concentrations used net secretion of calcium was observed. The s to m flux of the simultaneously measured paracellular marker mannitol at all calcium concentrations was remarkably higher than the m to s flux, resulting in net mannitol secretion. The results obtained from the calcium fluxes when clamping the transepithelial electrical potential agree well with those of the concentration dependence of the calcium fluxes: 1. Only m to s flux has a voltage independent, transcellular component. 2. Calcium s to m flux is totally voltage dependent, i.e. diffusive. 3. Diffusional s to m calcium flux is about 80% greater than the diffusional fraction of the m to s flux. Omitting glucose from the bathing solution effected a decrease of the transepithelial electrical potential and of the short circuit current by 91% and 85% respectively; net calcium secretion was almost abolished and net mannitol secretion remarkably reduced. Addition of glucose, which stimulates water absorption in the ileum as a metabolic substrate, activated m to s but significantly more pronounced s to m calcium flux parallel to that of mannitol. Dexamethasone, known to stimulate sodium and water absorption in the ileum by activation of the Na,K-ATPase, effected an increase of the transepithelial electrical potential difference and of the short circuit current by about 100% but had no influence on tissue resistance; m to s and more pronounced s to m calcium flux was stimulated after the induction by dexamethasone and net calcium secretion was increased by 70%. After pretreatment with 1,25-dihydroxyvitamin D3 tissue resistance was decreased by about 42%. The vitamin had no effect on net calcium or mannitol secretion but significantly increased bidirectional calcium and mannitol fluxes. Flux measurements in clamped preparations revealed: 1. 1,25-dihydroxyvitamin D₃ and dexamethasone has no effect on the cellular-mediated m to s calcium transport; 2. diffusive calcium flux in m to s and in the opposite direction, from s to m, is increased by the vitamin and by the hormone. In conclusion the net ileal calcium and mannitol secretion is the consequence of an asymmetry of the paracellular flux with a prevalence of the s to m flux over that in m to s direction. It is hypothesized that this prevalence is caused by an anomalous solvent drag effect. Paracellular calcium flux in both directions is increased by 1,25-dihydroxyvitamin D3 and dexamethasone by different mechanisms, as indicated by the changes in the electrical parameters of the tissue. Send offprint requests to U. Karbach at the above address     

Metabolism of 26,27-hexafluoro-1α,25-dihydroxyvitamin D3 and 26,27-hexafluoro1α,23(S)25-trihydroxyvitamin D3 in ROS17/2.8 cells transfected with a plasmid expressing CYP24

1. To clarify the possibility that the metabolism of 26,27-hexafl uoro-1α, 25-dihydroxyvitamin D₃ [F₆-1,25(OH)₂D₃] to 26,27-hexafluoro-1α,23(S),25-trihydroxyvitamin D₃ [F₆-1,23,25(OH)₃D₃ and that of F₆-1,23,25(OH)₃D₃ to 26,27-hexafluoro-23-oxo-1α,25-dihydroxyvitamin D₃ [F₆-23-oxo-1,25(OH)₂D₃] are catalysed by 25-hydroxyvitamin D 24-hydroxylase (CYP24), ROS17}2.8 cells transfected with a plasmid expressing CYP24 [pSVL-CYP24–] and a corresponding blank plasmid [pSLVCYP24R(®)] were used. 2. Incubation of [1β-³H]-F-1,25(OH)₂D₃ for 2 and 5 days with ROS17}2.8 cells transfected with pSVL-CYP24– generated a metabolite that co-migrated with authentic F₆ -1,23,25(OH)₃D₃ in both normal phase and reversed-phase HPLC systems. 3. Incubation of [1β-³H]-F₆ -1,23,25(OH)₃D₃ for 5 days with pSVL-CYP24– - transfected ROS 17}2.8 cells generated a metabolite that co-migrated with authentic F₆ -23-oxo-1,25(OH)₂D₃. In contrast, the metabolites F₆ -1,23,25(OH)₃D₃ or F₆ -23-oxo- 1,25(OH)₂D₃ were not generated in the cells transfected with pSVL-CYP24R(-). 4. The results indicate that CYP24 catalyses the conversion of F₆ -1,25(OH)₂D₃ to F₆ -1,23,25(OH)₃D₃ and that of F₆ -1,23,25(OH)₃D₃ to F₆ -23-oxo-1,25(OH)₂D₃.     

Human PXR-mediated induction of intestinal CYP3A4 attenuates 1α,25-dihydroxyvitamin D₃ function in human colon adenocarcinoma LS180 cells

Oxidative catabolism of 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)] is mediated by either CYP24A1 or CYP3A4. In this paper, we tested whether induction of CYP3A4 in the LS180 intestinal cell model enhances clearance of 1α,25(OH)(2)D(3) and blunts its hormonal effect on expression of the apical membrane calcium transport protein, TRPV6. Treatment with the hPXR agonist rifampin significantly increased CYP3A4 mRNA content and catalytic activity, but had no effect on CYP24A1 or TRPV6 mRNA content. Pre-treating cells with rifampin for 48h, prior to a 24h 1α,25(OH)(2)D(3) treatment phase, was associated with a subsequent 48% increase in the elimination of 1α,25(OH)(2)D(3) and a 35% reduction of peak TRPV6 mRNA. Introduction of the CYP3A4 inhibitor, 6',7'-dihydroxybergamottin, an active inhibitor in grapefruit juice, reversed the effects of rifampin on 1α,25(OH)(2)D(3) clearance and TRPV6 expression. Over-expression of hPXR in LS180 cells greatly enhanced the CYP3A4 responsiveness to rifampin pretreatment, and elicited a greater relative suppression of TRPV6 expression and an increase in 1α,25(OH)(2)D(3) disappearance rate, compared to vector expressed cells, following hormone administration. Together, these results suggest that induction of CYP3A4 in the intestinal epithelium by hPXR agonists can result in a greater metabolic clearance of 1α,25(OH)(2)D(3) and reduced effects of the hormone on the intestinal calcium absorption, which may contribute to an increased risk of drug-induced osteomalacia/osteoporosis in patients receiving chronic therapy with potent hPXR agonists. Moreover, ingestion of grapefruit juice in the at-risk patients could potentially prevent this adverse drug effect.     

Removal of the 26-methyl group from 19-nor-1α,25-dihydroxyvitamin D₃ markedly reduces in vivo calcemic activity without altering in vitro VDR binding, HL-60 cell differentiation, and transcription

Twelve new analogues of 19-nor-1α,25-dihydroxyvitamin D? (5-16) were prepared by convergent syntheses, employing the Wittig-Horner reaction. The necessary Grundmann type ketones (45-48), possessing fixed configurations of the hydroxyl group at C-25, were obtained by a multistep procedure from commercial vitamin D? and enantiomers of 1,3-butanediol (23 and 24). We have examined the influence of removal of one of the methyl groups located at C-25 on the biological in vitro and in vivo activity. The in vivo tests showed that the synthesized vitamin D compounds (5-16) exhibit reduced calcemic activity both in bone and in the intestine. However, in vitro potency of 2-methylene and 2α-methyl compounds (5-10, 13, and 14) remained similar or enhanced as compared to that of 1α,25-(OH)?D?.     

Comparative Effects of Doxercalciferol (1α-Hydroxyvitamin D2) Versus Calcitriol (1α,25-Dihydroxyvitamin D3) on the Expression of Transporters and Enzymes in the Rat In Vivo

Effects of 1.28 nmol/kg doxercalciferol [1α(OH)D₂], a synthetic vitamin D₂ analog that undergoes metabolic activation to 1α,25-dihydroxyvitamin D₂, the naturally occurring, biologically active form of vitamin D₂, on rat transporters and enzymes were compared with those of 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, active form of vitamin D₃; 4.8 and 6.4 nmol/kg] given on alternate days intraperitoneally for 8 days. Changes were mostly confined to the intestine and kidney where the vitamin D receptor (VDR) was highly expressed: increased intestinal Cyp24 and Cyp3a1 messenger RNA (mRNA) and a modest elevation of apical sodium-dependent bile salt transporter (Asbt) and P-glycoprotein (P-gp) protein; increased renal VDR, Cyp24, Cyp3a9, Mdr1a, and Asbt mRNA, as well as Asbt and P-gp protein expression; and decreased renal PepT1 and Oat1 mRNA expression. In comparison, 1α(OH)D₂ treatment exerted a greater effect than 1,25(OH)₂D₃ on Cyp3a and Cyp24 mRNA. However, the farnesoid X receptor -related repressive effects on liver Cyp7a1 were absent because intestinal Asbt, FGF15 and portal bile acid concentrations were unchanged. Rats on the alternate day regimen showed milder changes and lessened signs of hypercalcemia and weight loss compared with rats receiving daily injections (similar or greater amounts of 0.64–2.56 nmol/kg daily ×4) described in previous reports, showing that the protracted pretreatment regimen was associated with milder inductive and lesser toxic effects in vivo.     

α1L-Adrenoceptor mediation of smooth muscle contraction in rabbit bladder neck: a model for lower urinary tract tissues of man

1. The α₁-adrenoceptor population mediating contractile responses to noradrenaline (NA) in smooth muscles of the bladder neck from rabbit (RBN) has been characterized by use of quantitative receptor pharmacology.
2. Experiments with several `key'' α₁-adrenoceptor antagonists of varying subtype selectivities (RS-17053, BMY 7378, indoramin, 5-methylurapidil, prazosin, REC 15/2739, SNAP 5089, terazosin, WB 4101, tamsulosin, (+)-cyclazosin and RS-100329) were conducted. Schild regression analyses yielded affinity (mean pK_b) estimates of 7.1, 6.2, 8.6, 8.6, 8.4, 9.3, 7.0, 7.4, 8.9, 10.0, 7.1 and 9.3, respectively, although deviations from unit Schild regression slope question the robustness of data for RS-17053 and SNAP 5089.
3. The nature of antagonism by these agents and the profile of affinity determinations generated together suggest that a single α₁-adrenoceptor subtype mediates contractile responses of RBN to NA. Additional studies with phenylephrine indicated also an agonist-independence of this profile. Pharmacologically, this profile was reminiscent of that described as `α<sub>1L</sub>''-adrenoceptor, which has been shown to mediate contractions of several tissues including lower urinary tract (LUT) tissues of man. Furthermore, a similarity was noticed between the `α_1L''-adrenoceptor described here in RBN and the rabbit and human cloned α_1a-adrenoceptor (based on data from both whole cell radioligand binding at 37°C and [³H]-inositol phosphates accumulation assays), characterizations of which have been published elsewhere.
4. In conclusion, the RBN appears to provide a predictive pharmacological assay for the study of NA-induced smooth muscle contraction in LUT tissues of man.

     

Hyperosmotic Stimulus Down-regulates 1��, 25-dihydroxyvitamin D3-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System

The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1α, 25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1α,25(OH)₂D₃-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1α,25(OH)₂D₃-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1α,25(OH)₂D₃-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor κB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1α,25(OH)₂D₃. Knockdown of Runx2 inhibited 1α,25(OH)₂D₃-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1α,25(OH)₂D₃-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.     

3H-noradrenaline release from mouse iris–ciliary body: role of presynaptic muscarinic heteroreceptors

Sympathetic neurotransmitter release and its modulation by presynaptic muscarinic heteroreceptors were studied in mouse iris–ciliary bodies. Tissue preparations were preincubated with ³H-noradrenaline and then superfused and stimulated electrically. Firstly, experimental conditions were defined, allowing study of presynaptic sympathetic inhibition in mouse iris–ciliary body. If tissue was stimulated four times with 36 pulses/3 Hz, tritium overflow peaks were reliably and reproducibly measured. As expected, these stimulation conditions led to marked ₂-autoinhibition as indicated by the release-enhancing effect of the ₂-antagonists phentolamine and rauwolscine. To ensure autoinhibition-free ³H-noradrenaline release, which is optimal for studying presynaptic sympathetic inhibition, ₂-receptors were blocked in all subsequent experiments. Under these conditions, evoked tritium overflow was almost completely abolished in the presence of the sodium channel blocker tetrodotoxin, indicating a neuronal origin of ³H-noradrenaline release. Secondly, muscarinic inhibition of ³H-noradrenaline release was characterized using the conditions described above (36 pulses/3 Hz; phentolamine 1 M and rauwolscine 1 M throughout). The muscarinic receptor agonist oxotremorine M decreased evoked tritium overflow in a concentration-dependent manner with an IC₅₀ of 0.33 M and maximal inhibition of 51%. The concentration–response curve of oxotremorine M was shifted to the right by the muscarinic antagonists ipratropium and methoctramine, whereas pirenzepine was ineffective. The observed rank order of antagonist potencies, ipratropium > methoctramine > pirenzepine, which is typical for the M₂ subtype, indicates that presynaptic muscarinic receptors on sympathetic axons of mouse iris–ciliary bodies are predominantly M₂. Finally, inhibition of ³H-noradrenaline release by endogenously secreted acetylcholine was investigated. Longer pulse trains, 120 pulses/3 Hz and 600 pulses/5 Hz, were used and the cholinesterase inhibitor physostigmine was added to the superfusion medium to increase synaptic levels of endogenous acetylcholine. Under these conditions, ipratropium approximately doubled the evoked overflow of tritium, indicating that endogenously released acetylcholine can activate presynaptic muscarinic heteroreceptors. In conclusion, the present experiments establish measurement of the electrically induced release of ³H-noradrenaline from mouse iris–ciliary bodies. As in other species, noradrenaline release in this preparation was subject to presynaptic muscarinic inhibition. Our results also indicate that the presynaptic muscarinic receptors on sympathetic axons in mouse iris–ciliary body are predominantly M₂. Moreover, these receptors can be activated by both exogenous agonists and endogenously released acetylcholine and, hence, may operate physiologically in the interplay between the parasympathetic and sympathetic nervous system.     

Multi-variate approach to global protein aggregation behavior and kinetics: Effects of pH,NaCl, and temperature for α-chymotrypsinogen A

A global characterization of nonnative aggregation is presented for α-chymotrypsinogen A (aCgn) as a function of temperature (T), pH, and [NaCl]. Changes in unfolding free energy, native-state second osmotic virial coefficient (B₂₂), and aggregation pathways and kinetics were qualitatively and quantitatively determined using a combination of size-exclusion chromatography, multi-angle laser light scattering, and circular dichroism and fluorescence spectroscopy. Results were analyzed quantitatively using multi-variate statistical models and a recently developed mechanistic model that naturally accounts for changes in aggregation pathway due to competition between unfolding, nucleation, chain polymerization, aggregate condensation, and phase separation. State diagrams are presented that show the natural progression between different aggregation behaviors or pathways. Together, the results show that pH and [NaCl] determine both the rates of aggregation and what aggregation behavior or pathway holds. In contrast, T affects primarily only aggregation rates, in large part due to changes in unfolding free energy. Finally, it is shown that B₂₂ correlates strongly with which type of aggregation pathway is followed, suggesting a potentially useful approach for predicting and controlling physical properties of the resulting aggregates. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:645–662, 2010