RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers.

Individuals who have mutations in the breast-cancer-susceptibility gene BRCA1 (hereafter referred to as BRCA1-mutation carriers) frequently undergo prophylactic mastectomy to minimize their risk of breast cancer. The identification of an effective prevention therapy therefore remains a 'holy grail' for the field. Precancerous BRCA1(mut/+) tissue harbors an aberrant population of luminal progenitor cells, and deregulated progesterone signaling has been implicated in BRCA1-associated oncogenesis. Coupled with the findings that tumor necrosis factor superfamily member 11 (TNFSF11; also known as RANKL) is a key paracrine effector of progesterone signaling and that RANKL and its receptor TNFRSF11A (also known as RANK) contribute to mammary tumorigenesis, we investigated a role for this pathway in the pre-neoplastic phase of BRCA1-mutation carriers. We identified two subsets of luminal progenitors (RANK(+) and RANK(-)) in histologically normal tissue of BRCA1-mutation carriers and showed that RANK(+) cells are highly proliferative, have grossly aberrant DNA repair and bear a molecular signature similar to that of basal-like breast cancer. These data suggest that RANK(+) and not RANK(-) progenitors are a key target population in these women. Inhibition of RANKL signaling by treatment with denosumab in three-dimensional breast organoids derived from pre-neoplastic BRCA1(mut/+) tissue attenuated progesterone-induced proliferation. Notably, proliferation was markedly reduced in breast biopsies from BRCA1-mutation carriers who were treated with denosumab. Furthermore, inhibition of RANKL in a Brca1-deficient mouse model substantially curtailed mammary tumorigenesis. Taken together, these findings identify a targetable pathway in a putative cell-of-origin population in BRCA1-mutation carriers and implicate RANKL blockade as a promising strategy in the prevention of breast cancer.

Pharmacokinetics of tramadol and its primary metabolite O-desmethyltramadol in African penguins (Spheniscus demersus).

Analgesia is an important part of veterinary medicine, but until recently there have been limited studies on analgesic drugs in avian species. Tramadol represents an orally administered opioid drug that has shown analgesic potential in numerous species, including mammals, birds, and reptiles. The objective of this study was to determine the pharmacokinetic parameters of tramadol and its primary metabolite, O-desmethyltramadol (M1), after oral administration of tramadol hydrochloride (HCl) in African penguins (Spheniscus demersus). A dose of 10 mg/kg of tramadol HCl was administered orally to 15 birds, and blood was collected at various time points from 0 to 36 hr. Tramadol and M1 concentrations were determined and were consistent with therapeutic concentrations in humans through 12 hr in 9/15 birds for tramadol and 36 hr in 14/15 birds for M1. Based on these findings and a comparison with other avian studies, an oral dose of 10 mg/kg of tramadol once daily appears to be a promising analgesic option for African penguins.

Composition and stability of bacterial communities associated with granular activated carbon and anthracite filters in a pilot scale municipal drinking water treatment facility.

Granular activated carbon (GAC) is an alternative filter substrate for municipal water treatment as it provides a high surface area suitable for microbial colonization. The resulting microbial growth promotes biodegradation of organic materials and other contaminants from influent waters. Here, the community structure of the bacteria associated with three GAC and two anthracite filters was examined over 12 months to monitor changes in community composition. Nearly complete 16S rRNA genes were polymerase chain reaction amplified for terminal restriction fragment length polymorphism (T-RFLP) analyses. The identity of commonly occurring peaks was determined through the construction of five representative 16S rRNA clone libraries. Based on sequence analysis, the bacterial communities associated with both anthracite and GAC filters appear to be composed of environmentally derived bacteria, with no known human pathogens. Analysis of similarity tests revealed that significant differences in bacterial community structure occurred over time, with filter substrate playing an important role in determining community composition. GAC filters exhibited the greatest degree of bacterial community variability over the sampling period, while anthracite filters showed a lower degree of variability and less change in community composition. Thus, GAC may be a suitable biologically active filter substrate for the treatment of municipal drinking water.

Human paraoxonase 1 is the enzyme responsible for pilocarpine hydrolysis.

Pilocarpine has been widely used in ophthalmic preparations for the treatment of glaucoma and in oral preparations for the treatment of radiation-induced xerostomia and Sjögren syndrome. The major metabolic pathways of pilocarpine in human are hydrolysis and hydroxylation. It was found that CYP2A6 is responsible for the 3-hydroxylation, but the enzymes responsible for the hydrolysis have not been characterized. In this study, we attempted to identify esterases responsible for pilocarpine hydrolysis. Pilocarpine hydrolase activities in human liver microsomes and plasma were stimulated by the addition of CaCl(2), suggesting that the calcium-dependent esterase, paraoxonase (PON), was responsible for pilocarpine hydrolysis. To confirm this hypothesis, the pilocarpine hydrolase activity was measured using the recombinant human PONs (PON1, PON2, and PON3) established in this study, and the result was that only PON1 showed pilocarpine hydrolase activity. The effect of PON1 polymorphism (Q192R) on pilocarpine hydrolase activity was analyzed using recombinant human PON1 192Q and 192R and human plasma from 50 volunteers. The results showed that recombinant PON1 192R revealed significantly higher catalytic efficiency than PON1 192Q. In human plasma, the activity of the R/R genotype (117.0 ± 25.2 pmol · min(-1) · µl(-1), n = 23) was significantly higher than those of the Q/R and Q/Q genotypes (97.3 ± 21.0 pmol · min(-1) · µl(-1), n = 20 and 90.4 ± 26.2 pmol · min(-1) · µl(-1), n = 7, respectively). It is suggested that this polymorphism affects pilocarpine hydrolase activity. In this study, we found that human PON1 is the major enzyme for the catalytic efficiency of pilocarpine hydrolysis.

Effect of the administration of an oral hyaluronan formulation on clinical and biochemical parameters in young horses with osteochondrosis.

The aim of this study was to evaluate the clinical and biochemical effects of the administration of oral hyaluronan (Hyal-Joint [HJ]) on young horses with osteochondrosis (OC). Our hypotheses were that HJ administration is safe, would decrease the degree of synovial effusion and the concentration of nitric oxide (NO) and prostaglandin E2 (PGE2) in synovial fluid, and would increase the concentration of hyaluronic acid (HA) in plasma and synovial fluid. Eleven young horses with tarsocrural OC were included in a randomised, double-blinded, placebo-controlled pilot clinical trial. Six horses received 250 mg/day HJ for 60 days (T60) and five horses received a placebo. The initial values of the degree of synovial effusion, NO, PGE2 and HA concentrations in synovial fluid and HA concentration in plasma were obtained. The horses were evaluated in terms of the same parameters at the end of treatment (T60) and 30 days thereafter (T90). The differences between the groups for each of the parameters evaluated at T0, T60 and T90 were not significant. Nevertheless, the horses treated with HJ tended to show a lower score for synovial effusion as well as higher HA, NO and PGE2 concentrations in synovial fluid, but these differences were non-significant. At a dose of 250 mg/day, HJ did not produce any adverse clinical effects and was well tolerated by the horses.

Genetic polymorphisms of CYP2A6 affect the in-vivo pharmacokinetics of pilocarpine.

OBJECTIVE: Pilocarpine is metabolized to pilocarpic acid by plasma esterase and to 3-hydroxypilocarpine by CYP2A6. The objective of this study was to identify the determinants of interindividual differences in pilocarpine pharmacokinetics after oral administration and to investigate the possible involvement of genetic polymorphisms of CYP2A6. METHODS: The pharmacokinetic parameters of pilocarpine, pilocarpic acid, and 3-hydroxypilocarpine after oral administration of pilocarpine hydrochloride in 28 Japanese participants were calculated based on the data obtained from two phase-1 clinical studies. Probit analysis was carried out for the pharmacokinetic parameters, and participants were accordingly classified into two groups: poor metabolizers and nonpoor metabolizers of pilocarpine. The poor metabolizers were genotyped for CYP2A6, and the pharmacokinetic parameters in this group were compared with those in the nonpoor metabolizers. RESULTS: Relatively large interindividual differences were observed in the pharmacokinetic parameters of pilocarpine, pilocarpic acid, and 3-hydroxypilocarpine. Probit analysis of the pharmacokinetic parameters revealed that seven of the 28 participants exhibited significantly low plasma concentrations and urinary recovery of 3-hydroxypilocarpine and were classified as poor metabolizers. Genotyping analysis revealed that these poor metabolizers had two inactive CYP2A6 alleles, CYP2A6*4A, CYP2A6*7, CYP2A6*9, or CYP2A6*10. The apparent pilocarpine clearance was significantly lower in the poor metabolizers than in the nonpoor metabolizers (P<0.05). CONCLUSION: We demonstrated that CYP2A6 genotype is a contributor to pilocarpine pharmacokinetics, although the impact of the CYP2A6 polymorphism may be pharmacologically and toxicologically tolerable.

Evaluation of monolithic HPLC columns for various pharmaceutical separations: method transfer from conventional phases and batch to batch repeatability.

Methods developed on conventional particle-packed C18 columns for pilocarpine, propranolol, glibenclamide, glimepiride, insulin and their respective degradation products or related compounds were transferred from the conventional Superspher 100RP-18e column to Chromolith Performance RP-18e columns. All transfers were successful applying the same chromatographic conditions, except for insulin where the acetonitrile content of the mobile phase was reduced by 0.5%. The intraday and interday precisions for both retention time and peak area were evaluated over a wide concentration range. Results were found to be equal, or slightly better on Chromolith Performance with RSD%<1.1% in all cases. Monolithic batch to batch repeatability of both retention time and peak area, compared for monolithic columns from different batches gave an RSD% of less than 1.3%. The separation of each drug and its related products was investigated on monolithic columns at flow rates from 1 to 9 ml/min, and superior resolution was always obtained using monolithic over conventional columns at the same flow rate. A total of seven monolithic columns from four different batches were used in this study.

Involvement of CYP2A6 in the formation of a novel metabolite, 3-hydroxypilocarpine, from pilocarpine in human liver microsomes.

Pilocarpine is a cholinergic agonist that is metabolized to pilocarpic acid by serum esterase. In this study, we discovered a novel metabolite in human urine after the oral administration of pilocarpine hydrochloride, and we investigated the metabolic enzyme responsible for the metabolite formation. The structure of the metabolite was identified as 3-hydroxypilocarpine by liquid chromatography-tandem mass spectrometry and NMR analyses and by comparing to the authentic metabolite. To clarify the human cytochrome P450 (P450) responsible for the metabolite formation, in vitro experiments using P450 isoform-selective inhibitors, cDNA-expressed human P450s (Supersomes; CYP1A2, -2A6, -2B6, -2C9, -2C19, -2D6, -2E1, and -3A4), and liver microsomes from different donors were conducted. The formation of 3-hydroxypilocarpine in human liver microsomes was strongly inhibited (>90%) by 200 microM coumarin. Other selective inhibitors of CYP1A2 (furafylline and alpha-naphthoflavone), CYP2C9 (sulfaphenazole), CYP2C19 [(S)-mephenytoin], CYP2E1 (4-methylpyrazole), CYP2D6 (quinidine), and CYP3A4 (troleandomycin) had a weak inhibitory effect (<20%) on the formation. The highest formation activity was expressed by recombinant CYP2A6. The K(m) value for recombinant CYP2A6 was 3.1 microM, and this value is comparable with that of human liver microsomes (1.5 microM). The pilocarpine 3-hydroxylation activity was correlated with coumarin 7-hydroxylation activity in 16 human liver microsomes (r = 0.98). These data indicated that CYP2A6 is the main enzyme responsible for the 3-hydroxylation of pilocarpine. In conclusion, we identified a novel metabolite of pilocarpine, 3-hydroxypilocarpine, and we clarified the involvement of CYP2A6 in the formation of this molecule in human liver microsomes.

Evaluation of monolithic C18 HPLC columns for the fast analysis of pilocarpine hydrochloride in the presence of its degradation products.

Monolithic Performance C18 HPLC columns (Chromolith Performance RP-18e, Merck) were applied for the determination of pilocarpine hydrochloride in the presence of its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid. The method was validated using a set of six monolithic columns and compared to a conventional C18 column. The separation of pilocarpine from its degradation products was investigated on monolithic columns at different flow rates from 1 to 9 ml/min. Superior resolution was obtained using monolithic columns over the conventional C18 column at the same flow rate of 1 ml/min with a total run time of 9 min compared to 13.5 min for the conventional C18 column. Comparable resolution to that obtained in the C18 column (but with better peak symmetry) was obtained at a flow rate of 4 ml/min, although the total run time was reduced to 3.5 min. The precision for both retention time and peak area was investigated over a wide concentration range and found to be equal, or slightly better on Chromolith Performance compared to the conventional column. The overall RSDs% ranged from 0.5% to 1.16% for the conventional column, while for monolithic columns ranged from 0.38% to 0.87% at a flow rate of 1 ml/min and from 0.38% to 0.89% at a flow rate of 4 ml/min. Monolithic column to column reproducibility (n = 6) was measured. The RSDs% ranged from 0.34% to 0.68% for retention time and from 0.3% to 0.94% for peak areas. The detection and quantitation limits on monolithic columns at both flow rates (1 and 4 ml/min) were found to be 0.17 microg/ml and 0.5 microg/ml, compared to 0.31 microg/ml and 1 microg/ml on the conventional column. Monolithic silica rods have also shown the advantage of reducing the time to wash and to re-equilibrate the column. The method showed good linearity and recovery and was found to be suitable for the analysis of pilocarpine hydrochloride formulations.

Ontogenetic profile of ectonucleotidase activities from brain synaptosomes of pilocarpine-treated rats.

Adenosine, a well-known neuromodulator, can act as an endogenous anticonvulsant via the activation of adenosine A1 receptors. This adenine nucleoside can be produced in the synaptic cleft by the ectonucleotidase cascade, which includes the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5'-nucleotidase. It has been previously reported that ectonucleotidase activities are increased in female adult rats submitted to the pilocarpine model of epilepsy. Several studies have suggested that the immature brain is less vulnerable to morphologic and physiologic alterations after status epilepticus (SE). Here, we evaluate the ectonucleotidase activities of synaptosomes from the hippocampus and cerebral cortex of male and female rats at different ages (7-9, 14-16 and 27-30-day old) submitted to the pilocarpine model of epilepsy. Our results show that ATP and ADP hydrolysis in the hippocampus and cerebral cortex were not altered by the pilocarpine treatment in female and male rats at 7-9, 14-16 and 27-30 days. There were no changes in AMP hydrolysis in female and male rats submitted to the model at different ages, but a significant increase in AMP hydrolysis (71%) was observed in synaptosomes from the cerebral cortex of male rats at 27-30 days. Pilocarpine-treated male rats (60-70-day old) presented an enhancement in ectonucleotidase activities in the synaptosomes of the cerebral cortex (33, 40 and 64% for ATP, ADP and AMP hydrolysis, respectively) and hippocampus (55, 98 and 101% for ATP, ADP and AMP hydrolysis, respectively). These findings highlight differences between the purinergic system of young and adult rats submitted to the pilocarpine model of epilepsy.

Strain differences affect the induction of status epilepticus and seizure-induced morphological changes.

Genetic deficits have been discovered in human epilepsy, which lead to alteration of the balance between excitation and inhibition, and ultimately result in seizures. Rodents show similar genetic determinants of seizure induction. To test whether seizure-prone phenotypes exhibit increased seizure-related morphological changes, we compared two standard rat strains (Long-Evans hooded and Wistar) and two specially bred strains following status epilepticus. The special strains, namely the kindling-prone (FAST) and kindling-resistant (SLOW) strains, were selectively bred based on their amygdala kindling rate. Although the Wistar and Long-Evans hooded strains experienced similar amounts of seizure activity, Wistar rats showed greater mossy fiber sprouting and hilar neuronal loss than Long-Evans hooded rats. The mossy fiber system was affected differently in FAST and SLOW rats. FAST animals showed more mossy fiber granules in the naïve state, but were more resistant to seizure-induced mossy fiber sprouting than SLOW rats. These properties of the FAST strain are consistent with those observed in juvenile animals, further supporting the hypothesis that the FAST strain shares circuit properties similar to those seen in immature animals. Furthermore, the extent of mossy fiber sprouting was not well correlated with sensitivity to status epilepticus, but was positively correlated with the frequency of spontaneous recurrent seizures in the FAST rats only, suggesting a possible role for axonal sprouting in the development of spontaneous seizures in these animals. We conclude that genetic factors clearly affect seizure development and related morphological changes in both standard laboratory strains and the selectively bred seizure-prone and seizure-resistant strains.

Effects of status epilepticus on hippocampal GABAA receptors are age-dependent.

Long-term GABA(A) receptor alterations occur in hippocampal dentate granule neurons of rats that develop epilepsy after status epilepticus in adulthood. Hippocampal GABA(A) receptor expression undergoes marked reorganization during the postnatal period, however, and the effects of neonatal status epilepticus on subsequent GABA(A) receptor development are unknown. In the current study, we utilize single cell electrophysiology and antisense mRNA amplification to determine the effect of status-epilepticus induced by lithium-pilocarpine in postnatal day 10 rat pups on GABA(A) receptor subunit expression and function in hippocampal dentate granule neurons. We find that rats subjected to lithium-pilocarpine-induced status epilepticus at postnatal day 10 show long-term GABA(A) receptor changes including a two-fold increase in alpha1 subunit expression (compared with lithium-injected controls) and enhanced type I benzodiazepine augmentation that are opposite of those seen after status epilepticus in adulthood and may serve to enhance dentate gyrus inhibition. Further, unlike adult rats, postnatal day 10 rats subjected to status epilepticus do not become epileptic. These findings suggest age-dependent differences in the effects of status epilepticus on hippocampal GABA(A) receptors that could contribute to the selective resistance of the immature brain to epileptogenesis.

Correlated stage- and subfield-associated hippocampal gene expression patterns in experimental and human temporal lobe epilepsy.

Epileptic activity evokes profound alterations of hippocampal organization and function. Genomic responses may reflect immediate consequences of excitatory stimulation as well as sustained molecular processes related to neuronal plasticity and structural remodeling. Using oligonucleotide microarrays with 8799 sequences, we determined subregional gene expression profiles in rats subjected to pilocarpine-induced epilepsy (U34A arrays, Affymetrix, Santa Clara, CA, USA; P < 0.05, twofold change, n = 3 per stage). Patterns of gene expression corresponded to distinct stages of epilepsy development. The highest number of differentially expressed genes (dentate gyrus, approx. 400 genes and CA1, approx. 700 genes) was observed 3 days after status epilepticus. The majority of up-regulated genes was associated with mechanisms of cellular stress and injury - 14 days after status epilepticus, numerous transcription factors and genes linked to cytoskeletal and synaptic reorganization were differentially expressed and, in the stage of chronic spontaneous seizures, distinct changes were observed in the transcription of genes involved in various neurotransmission pathways and between animals with low vs. high seizure frequency. A number of genes (n = 18) differentially expressed during the chronic epileptic stage showed corresponding expression patterns in hippocampal subfields of patients with pharmacoresistant temporal lobe epilepsy (n = 5 temporal lobe epilepsy patients; U133A microarrays, Affymetrix; covering 22284 human sequences). These data provide novel insights into the molecular mechanisms of epileptogenesis and seizure-associated cellular and structural remodeling of the hippocampus.

Motor effects of GABA(A) antagonism in globus pallidus: studies of locomotion and tremulous jaw movements in rats.

RATIONALE: Although most rodent studies related to parkinsonian symptoms have focused on locomotion, tremulous jaw movements also have been used as a rodent model of tremor for investigating the circuitry of the basal ganglia. OBJECTIVE: There are multiple pathways involved in the generation of parkinsonian symptoms. The globus pallidus is a basal ganglia relay nucleus, and the present study was conducted to investigate the effect of pallidal GABA antagonism on locomotion and tremulous jaw movements. METHODS: Suppression of locomotion and induction of tremulous jaw movements were produced by repeated (i.e., 14 day) systemic administration of the dopamine D2 antagonist haloperidol, and by acute systemic injection of the muscarinic agonist pilocarpine. The GABA(A) antagonist bicuculline was injected into the globus pallidus, and its effects on locomotion in haloperidol- and pilocarpine-treated rats were assessed in the first group of experiments. In the second group of experiments, the effects of intrapallidal infusions of bicuculline on haloperidol- and pilocarpine-induced jaw movements were observed. RESULTS: Pallidal GABA antagonism stimulated locomotion when no other treatment was present, and also when animals were coadministered haloperidol or pilocarpine. Bicuculline suppressed haloperidol-induced jaw movements in a dose-related manner, and had no effect on pilocarpine-induced jaw movements. CONCLUSIONS: These results support the notion that there are distinct pathways conveying basal ganglia outflow and demonstrate that the striatopallidal pathway is involved in the generation of the haloperidol-induced tremulous jaw movements. These findings are consistent with some features of current models of basal ganglia function and may lead to an understanding of the specific mechanisms that generate parkinsonian symptoms.

Synthesis of chiral pilocarpine analogues via a C-8 ketone intermediate.

The synthesis of a chiral pilocarpine analogue 3 in which the lactone ring is replaced by an oxazolidinone and the bridging methylene group is in the ketone oxidation state has been accomplished. The utility of this compound as a key intermediate for the preparation of more complex structures was demonstrated by its reduction to two alcohol epimers and its reaction with a methylene ylide.

Miotic effect and irritation potential of pilocarpine prodrug incorporated into a submicron emulsion vehicle.

Pilocarpine prodrug, O,O'-dipivaloyl(1,2-ethylene) bispilocarpic acid diester, was introduced to a submicron emulsion vehicle in a dose equivalent to 0.5% pilocarpine base, and the formulation was studied in albino rabbits using miotic assay. Compared with pilocarpine HCl 0.5% solution delayed and prolonged miosis was observed after application of the prodrug emulsion. AUC(0-6 h) values for the prodrug emulsion and pilocarpine solution were 9252+/-1345 and 6845+/-1967%xmin, respectively. The prodrug was also administered twice daily for 5 days in the form of aqueous solution or submicron emulsion in order to study ocular irritation. Irritation potential of the prodrug was significantly reduced when submicron emulsion was used as a vehicle.

Determination of pilocarpic acid in human plasma by capillary gas chromatography with mass-selective detection.

A novel, highly sensitive method for the determination of pilocarpic acid (PA) in human plasma is described. In addition, the method provides for the conversion of the lactone, pilocarpine (P), to PA so that a total drug presence can be determined. Using novel high-performance liquid chromatographic conditions capable of separating P, isopilocarpine (I-P), PA and isopilocarpic acid (I-PA) from each other and from endogenous plasma impurities, it was confirmed that P exclusively and quantitatively converts to PA in heparinized human plasma during storage. For the determination of PA, the selective extraction of PA from protein-free plasma was accomplished using two different solid-phase extraction (SPE) cartridges in two consecutive SPE steps. After extraction, PA was lactonized with trifluoroacetic acid back to P, and both P and an internal standard were acylated using heptafluorobutyric anhydride (HFBA). The trifluoroacetylated derivatives were monitored using gas chromatography (GC) with mass spectrometric (MS) detection. This procedure allowed the sensitive and reliable determination of PA with a limit of quantification (LOQ) of 1 ng/ml, which could not be achieved using previously described methods. The assay was validated in the concentration range of 1 to 10 ng/ml with an intra-day precision (expressed as the coefficient of variation, C.V.) ranging from 9.9 to 0.5%. Inter-day precision for the quality control standard at 2.5 ng/ml showed a C.V. of 10.2%. Accuracy ranged from 94 to 102%. The assay was used to monitor the maximum systemic exposure to P, administered by the ocular route, in terms of total plasma PA (P and PA).

Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection.

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step -- ultrafiltration for plasma and dilution for urine samples -- followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.

Fractional factorial design optimization of the separation of pilocarpine and its degradation products by capillary electrophoresis.

The separation of pilocarpine and its degradation products by micellar electrokinetic capillary chromatography (MECC) has been optimized by using fractional factorial design of the experiments. Critical parameters were identified in a screening design, and an optimization design was used to optimize the separation. The optimal separation method was based on a borate buffer with sodium dodecyl sulfate (SDS). It is concluded that by using fractional factorial design it is possible to improve the separation of pilocarpine, its trans epimer, isopilocarpine and their hydrolysis products, pilocarpic acid and isopilocarpic acid.

Hydroxypropyl-beta-cyclodextrin increases the aqueous solubility and stability of pilocarpine prodrugs.

PURPOSE: The effects of 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on the aqueous solubility and stability of two lipophilic bispilocarpine prodrugs were investigated at pH 7.4. METHODS: The solubility of prodrugs was studied by phase-solubility method (0-72.5 mM HP-beta-CD). The stability of one of the prodrugs was investigated as a function of temperature (40 degrees C-70 degrees C) and HP-beta-CD concentration (0-72.5 mM). The apparent rate constants (k1, k2) for degradation of prodrug in 1:1 and 1:2 inclusion complexes and apparent stability constants (K1:1, K1:2) were calculated by the curve-fitting method. RESULTS: The phase-solubility diagrams were classified as Ap-type and the apparent stability constants (K1:1, K1:2) for 1:1- and 1:2-inclusion complexes were calculated to be 143-815 M-1 and 29-825 M-1, respectively. The stability of prodrug increased as a function of HP-beta-CD concentration over the studied temperature range. The shelf-life (t90%, calculated by the Arrhenius equation) of the prodrug in 72.5 mM HP-beta-CD solution increased 5.1-fold and 6.1-fold at 25 degrees C and 4 degrees C, respectively. CONCLUSIONS: The solubility of the prodrugs was shown to increase markedly in phase-solubility studies. The degradation rate of prodrug in stability studies was shown to be slower in the 1:2-complex than in the 1:1-complex and the relative amounts of complex species were found to be dependent on CD concentration.