Effects of formulation parameters on encapsulation efficiency and release behavior of thienorphine loaded PLGA microspheres.

To develop a long-acting injectable thienorphine biodegradable poly (d, l-lactide-co-glycolide) (PLGA) microsphere for the therapy of opioid addiction, the effects of formulation parameters on encapsulation efficiency and release behavior were studied. The thienorphine loaded PLGA microspheres were prepared by o/w solvent evaporation method and characterized by HPLC, SEM, laser particle size analysis, residual solvent content and sterility testing. The microspheres were sterilized by gamma irradiation (2.5 kGy). The results indicated that the morphology of the thienorphine PLGA microspheres presented a spherical shape with smooth surface, the particle size was distributed from 30.19 ± 1.17 to 59.15 ± 0.67 µm and the drug encapsulation efficiency was influenced by drug/polymer ratio, homogeneous rotation speed, PVA concentration in the water phase and the polymer concentration in the oil phase. These changes were also reflected in drug release. The plasma drug concentration vs. time profiles were relatively smooth for about 25 days after injection of the thienorphine loaded PLGA microspheres to beagle dogs. In vitro and in vivo correlation was established.

Formulation and evaluation of thienorphine hydrochloride sublingual delivery system.

Thienorphine hydrochloride (ThH) is a highly insoluble and readily metabolized partial-opioid agonist. It is used for the treatment of pain and heroin addiction. This study aimed to formulate and evaluate sublingual delivery systems containing ThH. Dimethyl-ß-cyclodextrin (DM-ß-CD) can enhance the solubility and permeability of hydrophobic drugs. In this paper, ThH cyclodextrin inclusion complexes were prepared and administrated sublingually with the objective of improving the drug's aqueous solubility, in vitro permeation rate, and in vivo absorption rate. The formulation was prepared with DM-ß-CD using the freeze-dried method and characterized using phase solubility, differential scanning calorimetry (DSC), X-ray and NMR analyses. The results of each test indicated the formation of dynamic inclusion complexes between ThH and DM-ß-CD. The inclusion complexes also showed significant increases in in vitro aqueous solubility and mucosal permeability. According to the pharmacokinetic study of the complex in rats, the AUC and C(max) values of the sublingual delivery group were 40 and 46 times higher than those of the gastrointestinal group, whereas t(max) was shorter, which proved that in vivo absorption and metabolism had been improved. It can therefore be concluded that the inclusion technology and sublingual delivery system were suitable for ThH development.

Effects of gamma-irradiation on PLGA microspheres loaded with thienorphine.

Ionizing radiation can be used as a drug sterilization technique, provided that the drug itself is not modified and that no toxic products are produced; moreover, if the irradiated product is a drug delivery system, its drug release characteristics must not be significantly altered by radiation. The aim of this work was to study the effects of sterilization by ionizing radiation on PLGA microspheres, containing thienorphine. Thienorphine PLGA microspheres were prepared by the O/W solvent evaporation method and characterized by HPLC, SEM and laser particle size analysis. Our experimental results showed that gamma-rays did not alter the drug content, and did not modify the kinetics of drug release from microspheres. Moreover, no significant changes in the shape and in the size distribution of microspheres were found after irradiation. In conclusion, the sterilization method is adequate because microspheres not underwent any change after exposition to gamma-irradiation.

[Feasibility of unilateral or bilateral nerve-sparing radical hysterectomy in patients with cervical cancer and evaluation of the post-surgery recovery of the bladder and rectal function].

OBJECTIVE: To investigate the feasibility of unilateral or bilateral nerve-sparing radical hysterectomy and evaluate the recovery of bladder and bowel function postoperatively. METHODS: From August 2008 to October 2009, sixty-one patients with cervical cancer stage Ib1 to IIa underwent radical hysterectomy (33 cases) and nerve-sparing radical hysterectomy (28 cases). Unilateral nerve-sparing radical hysterectomy was performed in 10 patients, and bilateral nerve-sparing radical hysterectomy (BNS) was performed in 18 patients. The data of operation time, blood loss, postoperative hospital stay days, residual urine volume, and postoperative complications were collected. The postoperative recovery of bladder and bowel function was evaluated. RESULTS: There were no significant differences between nerve-sparing radical hysterectomy (NSRH) and radical hysterectomy (RH) groups in operation time [NSRH: (224.5 ± 40.0) min, RH: (176.4 ± 30.0 min)], blood loss [NSRH: (464.3 ± 144.0) ml, RH: (374.2 ± 138.7) ml], postoperative hospital stay days [NSRH: (8.4 ± 2.0) d, RH: (9.2 ± 1.8) d, and residual urine volume [NSRH: (64.8 ± 16.9) ml, RH: (70.6 ± 16.0) ml]. There were also no significant differences between UNSRH and BNSRH groups in operation time [UNSRH: (208.5 ± 28.5) min, BNSRH: (233.3 ± 43.1) min], blood loss [UNSRH: (440.0 ± 104.9) ml, BNSRH: (477.8 ± 162.90) ml], postoperative hospital stay days [UNSRH: 9.1 ± 1.8) d, BNSRH: (8.7 ± 2.1 d], and the residual urine volume [UNSRH: (68.3 ± 12.5) ml, BNSRH: (62.8 ± 20.0) ml]. There was a significant difference in the time of the Foley catheter removal between NSRH [(12.4 ± 5.2) d] and RH [(22.4 ± 9.7) d] groups. There was a significant difference in the time of the Foley catheter removal between UNSRH [(18.2 ± 3.6) d] and BNSRH [(9.1 ± 2.0) d] groups. During the postoperative 3 weeks follow-up, the patients in the NSRH group had a higher rate of satisfaction at urination and defecation (100%, 75%) than the RH group (54.5%, 24.2%). CONCLUSION: UNSRH and BNSRH are safe and feasible techniques for early stage cervical cancer, and may significantly improve the recovery of bladder and rectal function.

Secular Trends in the Incidence of and Mortality Due to Alzheimer's Disease and Other Forms of Dementia in China From 1990 to 2019: An Age-Period-Cohort Study and Joinpoint Analysis.

BACKGROUND: The number of individuals with dementia is increasing, which negatively affects families, communities, and health care systems worldwide. The changes in the incidence of and mortality due to Alzheimer's disease and other forms of dementia at the national level in China have remained unknown over the past three decades. METHODS: Following the general analytical strategy used in the Global Burden of Disease Study (GBD) 2019, the age- and sex-specific incidence and mortality rates for dementia in China were analyzed. Trends in the incidence of and mortality due to dementia from 1990 to 2019 were assessed by joinpoint regression analysis. The effects of age, period and cohort on the incidence of and mortality due to dementia were estimated by an age-period-cohort model. RESULTS: The age-standardized incidence and mortality rates per 100,000 population were 103.83 (95% UI, 87.93-118.87) and 23.32 (95% UI, 5.66-61.31), respectively, for dementia in 2019. From 1990 to 2019, a significant average annual percentage change (AAPC) in the age-standardized incidence rate was observed in both males [0.49% (95% CI, 0.43-0.55%)] and females [0.31% (95% CI, 0.24-0.38%)], and the age-standardized mortality rate significantly increased in males [0.42% (95% CI, 0.31-0.53%)]. The population aged 55-59 years had the highest AAPC in the incidence of dementia [0.87% (95% CI, 0.81-0.93%)]. The age effect showed that the relative risks (RRs) of incident dementia and dementia-associated mortality increased with age among males and females, and individuals aged 60 years and older had significantly higher RRs. The RR of incident dementia increased with time, and the RR started to substantially increase in 2009. The cohort effect showed that the incidence decreased in successive birth cohorts. CONCLUSION: Alzheimer's disease and other forms of dementia continue to become more common among males and females in China, and the associated mortality rate in males significantly increased from 1990 to 2019. Early interventions should be implemented to reduce the burden of dementia on individuals at high risk in China.

Preparation and in vivo evaluation of thienorphine-loaded PLGA microspheres.

Thienorphine-loaded microspheres composed of poly(D,L-lactide-co-glycolide) were prepared by an O/W emulsion solvent evaporation method. HPLC was used to determine the drug loading and drug release, while a LC-MS-MS system was employed to analyze the plasma drug concentration. Results indicated that the PLGA particles obtained were spherical and of appropriate size. The formulation was stable during the test period. In vitro drug release from the microspheres was sustained for about 28 days mostly by the diffusion mechanism. The plasma drug concentration-time profiles were relatively smooth for about 28 days after subcutaneous injection of the drug-loaded microspheres to rats, compared with that for drug suspension. In vitro and in vivo correlation was established.

[Clinical analysis of 13 cases with vaginal intraepithelial neoplasia].

OBJECTIVE: To investigate the pathogenesis, high risk factors, clinical characteristics, methods of diagnosis and treatment, and prognosis of vaginal intraepithelial neoplasia (VAIN). METHODS: The clinical data of thirteen cases of VAIN treated in Zhejiang Provincial Cancer Hospital dated Mar. 2002 through Dec. 2008 were reviewed and analyzed retrospectively. RESULTS: Twelve of 13 VAIN cases were performed the human papillomavirus (HPV) detection with 92% (11/12) HPV positive rate. None of the cases shown specific clinical manifestation. Among the 13 cases, 6 of them accompanied with cervical cancer, 4 cases with cervical intraepithelial neoplasia (CIN), and 3 cases with vulvar intraepithelial neoplasma (VIN). Five cases synchronously diagnosed with cervical lesion and 3 with vulva lesion were underwent surgery, while the other 5 cases were diagnosed metachronously. Among 8 cases underwent surgery, 1 case with CIN underwent argon plasma coagulation (APC) after surgery, 1 case with the positive edge of VIN underwent APC. During follow up, 1 case with locally advanced cervical cancer underwent radiotherapy again, 3 cases with VAIN received APC, while 1 cervical cancer cases with VAIN received no treatment. The average follow-up time was 25.6 months (range 6-87 months). Two cases died of cervical cancer metastasis. The other 11 cases were normal and still alive. None of them progressed to invasive carcinoma. CONCLUSIONS: The main reason of VAIN is HPV infection. There are not specific clinical manifestations, usually diagnosed when reviewing cervical or vulva lesions and rarely progressed to invasive carcinoma. The main treatment of VAIN is surgery with the adjuvant treatment of APC.

Osteogenesis mechanism of chitosan-coated calcium sulfate pellets on the restoration of segmental bone defects.

A radial segmental defect model of a rabbit was used to study the restoration effect on defects treated with chitosan-coated pressed calcium sulfate pellets combined with recombinant human bone morphogenetic protein-2 (rhBMP-2), coated pressed calcium sulfate pellets, and uncoated pressed calcium sulfate pellets. Nothing was implanted in the control group. After 4, 8, and 12 weeks, the results indicated that coated pressed calcium sulfate pellets combined with rhBMP-2 and coated pressed calcium sulfate pellets facilitated new bone formation on defected bones and that, particularly, the coated pressed calcium sulfate pellets combined with rhBMP-2 was more effective than the coated pressed calcium sulfate pellet. Histologic and tetracycline fluorimetric findings showed that the osteogenesis mechanism of chitosan-coated pressed calcium sulfate pellets is membrane bone formation, and the pellets showed slightly slower resorption that closely coincides with the growth rate of new bone.

[Analysis of therapeutic result and prognostic factor in primary fallopian tube carcinoma].

OBJECTIVE: To investigate the impact of treatment modality and clinicopathologic profile on prognosis in primary fallopian tube carcinoma. METHODS: The data of 64 cases with primary fallopian tube carcinoma treated between January 1991 and June 2006 were analyzed. The clinicopathological data were retrospectively analyzed. RESULTS: The overall 5-year survival rate of this series was 56.3%. The overall 3- and 5-year survival rate was 84.6% and 65.4% in surgical staging group versus 58.3% and 33.3% in no surgical staging group with a significant difference between two groups (P = 0.0429; P = 0.043), which was 89.5% and 68.4% in optimal cytoreduction group versus 66.7% and 41.7% in suboptimal cytoreduction group (P = 0.0466; P = 0.0444). However, there was no significant difference in 3-year and 5-year survival rate between the group with pelvic lymphadenectomy and the group without (84.2% vs. 69.2%, P = 0.4667; 63.1% vs. 53.8%, P = 0.459), and also between the group treated using CAP/CP regimen and the group by TP regimen for chemotherapy (81.8% vs. 80.0%, P = 0.8946; 59.1% vs. 60.0% P = 0.9582). It was found that the 5-year survival was correlated with FIGO stage (III-IV vs. I - II, P = 0.0197), differentiation grade (G3 vs. G1 + G2, P = 0.003), pathologic type (other type vs. serous, P = 0.0494), lymph nodes status (positive vs. negative, P = 0.0295). CONCLUSION: Surgical staging, optimal cytoreduction, differentiation grade, pathologic type, lymph node status are important factors influencing the 5-year survival in primary fallopian tube carcinoma. Pelvic lymphadenectomy is necessary and feasible to perform during the procedure of surgical staging and cytoreduction. CAP/CP and TP regiment are similarly effective in adjuvant chemotherapy for primary fallopian tube carcinoma.

Pharmacodynamic comparison of prostaglandin E1 administered by different routes to rats.

The pharmacodynamics of prostaglandin E1 (PGE1) administered by different routes to rats was investigated in this paper. The hypotensive effect of PGE, was used as an index of drug efficacy, pharmacodynamic parameters such as time to reach peak effect (Tmax), maximal percentage of blood pressure decrease (Emax, %), duration of effect (Td), and the area under the blood pressure decrease percent-time curves (AUC, % x min) were determined after PGE1 given to rats intranasally, sublingually, intraperitoneally (ip), and intramuscularly (im), separately, and compared with those obtained from intravenous (iv) administration. Similar to iv route, the pharmacodynamic parameters of PGE1 from the other administration routes, Emax, Td and in particular AUC values were all increased with increasing doses, showing dose-efficacy relationship. Tmax was found to be approximately 3-4 min for nasal route, 3-8 min for im, 6-8 min for ip and 12-30 min for sublingual route, separately. Thus, the order of magnitude of absorption rate of the drug was as follows: nasal approximately = im > ip > sublingual. If the pharmacological bioavailability (PF) for each administration route was used as a tentative measure of drug absorption extent, the order of magnitude of absolute bioavailability appeared as follows: nasal > im approximately = ip > sublingual. Furthermore, the interindividual difference was found to be larger for im and ip route than that for nasal and sublingual route. These results indicate nasal and sublingual routes are two promising routes for the systemic delivery of PGE1 in clinical applications.

Study on brain targeting of raltitrexed following intranasal administration in rats.

PURPOSE: To investigate the levels of raltitrexed (RTX) in blood and different brain tissues in rats and to find out whether there is any direct drug transport from nasal cavity to brain tissues following intranasal (i.n.) administration. METHODS: Raltitrexed was administered to male Sprague-Dawley rats either intranasally or intravenously. Drug concentrations in blood and brain tissues were determined at different times post dosing. RESULTS: The plasma levels achieved after i.n. administration were significantly lower than those following intravenous (i.v.) administration (P < 0.05) before 120 min; but were significantly higher (P < 0.05) after 120 min. Following i.n. administration, RTX concentrations in different brain tissues were constantly detected for quite a long time and differed significantly from each other, the rank order being C (OB) > C (OT) > C (CR) > C (CL). On the contrary, RTX appeared only at the initial two or three time points in different brain regions after i.v. injection, and the concentrations were similar. AUC values in four brain regions by the nasal route were 54- to 121-fold compared with the i.v. route, the drug targeting index (DTI) values of nasal route were 71-158 for different brain regions, and about 99% of RTX content within 360 min in the brain were transported via the olfactory pathway. CONCLUSIONS: These results showed that antineoplastic RTX could be directly transported into the brain via the olfactory pathway in rats.

[Combined effect of cosolvent and cyclodextrin on solubilization of insoluble drugs].

AIM: To investigate the combined effect of cosolvent and cyclodextrin (CD) on solubilization of insoluble drugs. METHODS: Phase-solubility method was applied to determine solubilization of two diterpenoids in cosolvent / cyclodextrin combinations. The combined effect was evaluated and explained with an established mathematical model, and the model parameters were calculated by means of nonlinear regression analysis. RESULTS: The strong agreement between the predicted and the observed solubility data supports the validity of the proposed model, with the determination coefficients of two regression models were 0.993 and 0.992, separately. CONCLUSION: The validated mathematical model can be used to explain and predict the combined solubilization of the two insoluble drugs in different cosolvent systems.

[Effect of preparation technique on in vitro release mechanism of huperzine A microspheres].

AIM: To investigate the effect of preparation technique on in vitro release mechanism of huperzine A-PLGA microspheres. METHODS: Huperzine A-PLGA microspheres were prepared by two kinds of O/O emulsion solvent evaporation method (method A and B). In vitro release mechanism was explained by release profile, degradation rate and swelling rate of microspheres in vitro. The microspheres morphology and drug distribution within microspheres were observed in order to explain further the drug release mechanism. RESULTS: The encapsulation efficiency of huperzine A microspheres prepared by method A and B was 47.60% and 83.50% respectively. Microspheres prepared by method A could sustain release for 35 days with nearly no initial burst release. The release profile fitted well to zero order equation and drug release mainly through degradation and diffusion mechanism. Huperzine A microspheres prepared by method B could sustain release for 21 days with some evidence of initial burst release. The release profile fitted well to the Higuchi equation and drug release was mainly through diffusion mechanism. CONCLUSION: Huperzine A microspheres prepared by method A had more desirable release profile.

Preparation of prostaglandin E1-hydroxypropyl-beta-cyclodextrin complex and its nasal delivery in rats.

The potential use of hydroxypropyl-beta-cyclodextrin (HP-betaCD) in the solubilization and stabilization of prostaglandin E(1) (PGE(1)) was investigated. The solubility and chemical stability of PGE(1) were significantly improved upon complexation with HP-betaCD. The nasal delivery of PGE(1) from the complex formulation was also studied in Wistar rats and compared with intravenous administration. PGE(1) complex after nasal administration caused a rapid decrease of blood pressure and exhibited an obvious dose-efficacy relationship, showing results nearly similar to those obtained for intravenous route. The time to reach the peak effect (T(max)) was approximately 3-4 min. Except T(max), other pharmacodynamic parameter values such as the maximal percent of blood pressure decrease (E(max), %), the lasting time of effect (T(d)), and the area under the curve (AUC, blood pressure decrease % min) were increased with increasing the administered doses. The E(max), T(d), and in particular AUC values between doses were significantly different (P < 0.01), but T(max) between doses were not significantly different (P < 0.05). The AUC values per unit dose of PGE(1) for nasal administration, however, were smaller than those for intravenous route, probably due to the incomplete absorption of nasally administered PGE(1). Besides, the in vitro effect of the PGE(1) complex on nasal mucociliary movement was also investigated with a toad palate model. The PGE(1) complex formulation exerted only minor effect on nasal mucociliary movement. These results indicate that the PGE(1)-HP-betaCD complex formulation for nasal delivery is a very promising preparation with advantages such as rapid and effective absorption, good chemical stability, ease of administration, and minor nasal ciliotoxicity.

Preparation and in vivo evaluation of huperzine A-loaded PLGA microspheres.

Huperzine A-loaded microspheres composed of poly(D,L-lactide-co-glycolide) were prepared by an ONV emulsion solvent evaporation method. The characterization of the microspheres such as drug loading, size, shape and release profile was described. The in vitro release in the initial 7 days was nearly linear with 10% released per day. Thereafter drug release rate became slow gradually and about 90% drug released at day 21. The in vitro release rate determined by dialysis bag method had a good correlation with the in vivo release rate. Huperzine A aqueous solution was intramuscularly injected (i.m.) at 0.4 mg/kg and microspheres were intramuscularly injected at 8.4 mg eq huperzine A/kg in rats. The maxium plasma concentration (Cmax ) after i.m. microspheres was only 32% of that after i.m. solution. Drug in plasma could be detected until day 14 and about 5% of administered dose was residued at the injection site at day 14. The relative bioavailability of huperzine A microspheres over a period of 14 days was 94.7%. Inhibition of acyecholinesterase activity (AchE) in rat's cortex, hippocampus and striatum could sustain for about 14 days. In conclusion, huperzine A-loaded microspheres possessed a prolonged and complete drug release with significant inhibition of AchE for 2 weeks in rats.

Clinical analysis of twenty cases of cervical cancer associated with pregnancy.

OBJECTIVES: The objectives are to investigate the therapeutic methods and prognosis of cervical cancer associated with pregnancy. METHODS: Combined with a literature review, we analyzed the clinical characteristics, diagnosis and treating process, and prognosis of 20 cases of cervical cancer associated with pregnancy admitted in our hospital from 2000 to 2009. RESULTS: Fifteen cases were treated with surgery, four cases with radiotherapy or synchronous chemoradiation, and one case with palliative systemic chemotherapy. Fourteen cases were disease-free survivals, five cases died of distant metastasis, and one case was lost to follow-up. Also, for survival, there were seven in eight cases of early pregnancy, two in four cases of mid-pregnancy, one in two cases of late pregnancy, four in six postpartum cases, and one in three cases of vaginal delivery. CONCLUSIONS: There was a close relationship between clinical stage, diagnosis time, delivery method, and the prognosis of cervical cancer associated with pregnancy. For late pregnancy and postpartum cervical cancer surgery, preoperative and postoperative combination chemotherapy might improve the prognosis.

Treatment and prognosis of cervical cancer associated with pregnancy: analysis of 20 cases from a Chinese tumor institution.

This study was designed to investigate the therapeutic approaches and prognosis for cervical cancer associated with pregnancy. Clinical information, therapeutic strategies, and follow-up results of 20 patients with cervical cancer associated with pregnancy from Jan. 2000 to June 2009 in the Zhejiang Cancer Hospital were retrospectively analyzed. The International Federation of Gynecology and Obstetrics (FIGO) stages were: in situ (n=1), stage IA1 (n=1), stage IB1 (n=5), stage IB2 (n=1), stage IIA (n=8), stage IIB (n=3), and stage IIIB (n=1). Eight patients were in the first trimester of pregnancy, four in the second, two in the third, and six at postpartum when diagnosed. The therapeutic strategies were either single or combined modalities, including surgery, radiotherapy, and chemotherapy. Fourteen patients survived, five patients died (four of remote metastasis and one of uremia), and one patient was lost to follow-up. One newborn from a patient at stage IIA carcinoma in the third trimester with postponed therapy six weeks after diagnosis survived. Retarded fetal growth was observed in one patient receiving neoadjuvant chemotherapy and cesarean section. Out of the six postpartum patients, three underwent cesarean section and survived, whereas only one out of the three who underwent vaginal delivery survived. The remaining two died of remote metastasis. Therefore, personalized treatment is necessary for cervical cancer associated with pregnancy. Cervical cancer patients in the third trimester of pregnancy can continue the pregnancy for a short period of time. There may be potential risk for the fetus by chemotherapy during pregnancy. Cesarean section is the preferred mode of delivery for pregnant cervical cancer patients.

The Role of p21 in Apoptosis, Proliferation, Cell Cycle Arrest, and Antioxidant Activity in UVB-Irradiated Human HaCaT Keratinocytes.

BACKGROUND: Skin cancer is the most common cancer in the United States, and ultraviolet B (UVB) radiation-induced DNA damage is the major environmental factor underlying skin cancer development. p21, a p53-inducible protein, plays a key role in the cellular response to UVB-induced DNA damage. MATERIAL AND METHODS: Through p21 silencing and overexpression, we investigated the role of p21 in apoptosis, proliferation, cell cycle arrest, and oxidative stress in UVB-irradiated HaCaT keratinocytes. RESULTS: We found that UVB exposure induced significant p21 downregulation (p<0.05) and was associated with significantly increased apoptosis, significantly decreased proliferation, and significantly increased G2 phase arrest (p<0.05) in UVB-irradiated HaCaT keratinocytes. p21 silencing significantly promoted apoptosis, significantly inhibited G2 phase arrest, and significantly inhibited proliferation (p<0.05), but after UVB irradiation, p21 silencing demonstrated a less significant pro-apoptotic effect and a more significant inhibition of G2 phase arrest (p<0.05), which was reflected in significantly higher proliferative activity (p<0.05). p21 overexpression acted in an anti-apoptotic manner in the absence of UVB-induced DNA damage but acted in a pro-apoptotic manner in the presence of UVB-induced DNA damage, displaying an "antagonistic duality" similar to other growth-promoting oncoproteins. p53 expression mirrored p21 expression, suggesting a regulatory feedback mechanism between p21 and p53 expression. p21 overexpression significantly downregulated glutathione peroxidase and superoxide dismutase antioxidant activity (p<0.05) while significantly upregulating hydrogen peroxide and malondialdehyde content (p<0.05), suggesting a role in decreasing antioxidant defense capabilities in UVB-irradiated HaCaT keratinocytes. CONCLUSIONS: These findings reveal that p21 may play a key role in HaCaT keratinocytes' response to UVB exposure.