Invirase 200 mg

Concomitant use of INVIRASE with drugs that are substrates of P-gp may lead to elevated plasma concentrations of the concomitant drugs. Monitoring for toxicity is therefore recommended. Compounds that are substrates of P-gp include cyclosporine, paclitaxel, and vinblastine.

Garlic capsules should not be used when taking saquinavir without ritonavir due to the risk of decreased saquinavir plasma concentrations. No data are available for the coadministration of INVIRASE/ritonavir and garlic capsules.

Coadministration of 600 mg of saquinavir soft gel capsules and quadruple strength grapefruit juice as a single administration in healthy volunteers resulted in a 54% increase in exposure to saquinavir.

For patients with CL_CR 30 to 60 mL/min the dose of clarithromycin should be reduced by 50%.

Compounds that are substrates of CYP3A4 (e.g. alfentanyl, alprazolam, amiodarone, calcium channel blockers, clindamycin, carbamazepine, cyclosporine, dapsone, disopyramide, fentanyl, nefazodone, pimozide, quinidine, quinine, tacrolimus, warfarin) may have elevated plasma concentrations when coadministered with INVIRASE; therefore, these combinations should be used with caution and patients should be monitored for toxicities associated with such drugs.

Since INVIRASE is coadministered with ritonavir, the ritonavir product monograph should be reviewed for additional drugs that should not be coadministered.

Coadministration with compounds that are potent inducers of CYP3A4 (e.g., phenobarbital, phenytoin, dexamethasone, carbamazepine) may result in decreased plasma levels of saquinavir.

In a study investigating the drug-drug interaction of rifampin 600 mg/day daily and INVIRASE 1000 mg/ritonavir 100 mg twice daily (ritonavir-boosted INVIRASE) involving 28 healthy volunteers, 11 of 17 healthy volunteers (65%) exposed concomitantly to rifampin and ritonavir-boosted INVIRASE developed severe hepatocellular toxicity presented as increased hepatic transaminases. In some subjects, transaminases increased up to >20-fold the upper limit of normal and were associated with gastrointestinal symptoms including abdominal pain, gastritis, nausea, and vomiting. Following discontinuation of all three drugs, clinical symptoms abated and the increased hepatic transaminases normalized (see Contraindications).

Concomitant use of INVIRASE and St John’s Wort or products containing St. John’s Wort is not recommended due to risk of decreased plasma concentrations of saquinavir which may lead to loss of virologic response and possible resistance to saquinavir or to the class of protease inhibitors.

Several drug interaction studies have been completed with both INVIRASE and saquinavir soft gel capsules. Observations from drug interaction studies with saquinavir soft gel capsules may not be predictive for INVIRASE. Because ritonavir is coadministered, prescribers should refer to the prescribing information for ritonavir regarding drug interactions associated with this drug.

The metabolism of saquinavir is mediated by cytochrome P450, with the specific isoenzyme CYP3A4 responsible for 90% of the hepatic metabolism. Additionally, saquinavir is a substrate for P-Glycoprotein (Pgp). Therefore, drugs that affect CYP3A4 and/or Pgp, may modify the pharmacokinetics of saquinavir. Similarly, saquinavir might also modify the pharmacokinetics of other drugs that are substrates for CYP3A4 or Pgp.

INVIRASE (saquinavir mesylate) should only be used in combination with ritonavir, because it significantly inhibits saquinavir’s metabolism to provide increased plasma saquinavir levels.

For serious toxicities that may be associated with saquinavir, the drug should be interrupted. INVIRASE at doses less than 1000 mg with 100 mg ritonavir bid are not recommended since lower doses have not shown antiviral activity. For recipients of combination therapy with INVIRASE and ritonavir, dose adjustments may be necessary. These adjustments should be based on the known toxicity profile of the individual agent and the pharmacokinetic interaction between saquinavir and the coadministered drug (see Drug Interactions). Physicians should refer to the Product Monographs of these drugs for comprehensive dose adjustment recommendations and drug-associated adverse reactions.

When administered with lopinavir/ritonavir 400/100 mg bid, the appropriate dose of INVIRASE is 1000 mg bid (with no additional ritonavir).

INVIRASE should only be used in combination with ritonavir. The recommended dosage regimen is INVIRASE 1000 mg (5×200 mg capsules or 2×500 mg tablets) two times daily with ritonavir 100 mg two times daily, in combination with other antiretroviral agents. Ritonavir should be taken at the same time as INVIRASE, and within 2 hours after a meal.

For adults or adolescents over the age of 16 years unable to take INVIRASE 500 mg film-coated tablets, INVIRASE should be given in the form of 200 mg capsules.

Ritonavir should be taken at the same time as INVIRASE, and within 2 hours after a meal. INVIRASE capsules and tablets should be swallowed unchewed, with water or some other non-alcoholic drink. You should avoid excessive consumption of alcohol during your treatment with INVIRASE.

The missed dose should be taken as soon as it is remembered, then the regular dosing schedule should be continued. Two doses should not be taken at the same time.

Additionally, the following adverse experiences of any intensity, at least remotely related to saquinavir, reported in clinical trials using INVIRASE or saquinavir soft gel capsules with or without ritonavir, and not mentioned in Table 2 are provided for completeness and are listed below by body system:

Serious and non-serious adverse events from post-marketing spontaneous reports (where saquinavir was taken as the sole protease inhibitor or in combination with ritonavir), for which a causal relationship to saquinavir cannot be excluded are listed below. As these data come from the spontaneous reporting system, the frequency of adverse reactions is unknown.

• Immune System Disorders: allergic reactions and hypersensitivity.

  
  

• Metabolism and Nutrition Disorders:

  
  

  • diabetes mellitus or hyperglycemia sometimes associated with ketoacidosis (see Warnings and Precautions, Endocrine and Metabolism, Diabetes Mellitus and Hyperglycemia).

    
    

  • Lipodystrophy: Combination antiretroviral therapy has been associated with redistribution of body fat (lipodystrophy) in HIV infected patients including the loss of peripheral and facial subcutaneous fat, increased intra-abdominal and visceral fat, breast hypertrophy and dorsicervical fat accumulation (buffalo hump) (see Warnings and Precautions, Body as a Whole, Fat Redistribution).

    
    

  • Combination antiretroviral therapy has been associated with metabolic abnormalities such as hypertriglyceridemia, hypercholesterolemia, insulin resistance, hyperglycemia and hyperlactatemia (see Warnings and Precautions).

    
    

• Nervous System Disorders: somnolence, convulsions.

  
  

• Vascular Disorders:

  
  

  • thrombophlebitis.

    
    

  • There have been reports of increased bleeding, including spontaneous skin hematomas and hemarthroses, in hemophilic patients type A and B treated with protease inhibitors (see Warnings and Precautions, Hematologic, Hemophiliac Patients).

    
    

• Hepatobiliary Disorders: isolated elevation of transaminases and hepatitis.

  
  

Additional adverse events that have been observed during the postmarketing period are similar to those seen in clinical trials with INVIRASE and saquinavir soft gel capsules alone or in combination with ritonavir.

appetite decreased, weight increase, dehydration.

The clinical trial database for INVIRASE consists of >6000 patients, with over 100 patients followed for >2 years. Limited experience is available from three single-dose studies investigating the pharmacokinetics of the INVIRASE 500 mg film coated tablet compared to the INVIRASE 200 mg capsule in healthy volunteers (n=140). In two of these studies saquinavir was boosted with ritonavir; in the other study, saquinavir was administered as single drug. The INVIRASE tablet and the capsule formulations were similarly tolerated. The most common adverse events were gastrointestinal disorders (such as diarrhea). Since bioequivalence was demonstrated, no difference in safety profile is expected between the two formulations of INVIRASE.

Because clinical trials are conducted under very specific conditions the adverse reaction rates observed in the clinical trials may not reflect the rates observed in practice and should not be compared to the rates in the clinical trials of another drug. Adverse drug reaction information from clinical trials is useful for identifying drug-related adverse events and for approximating rates.

The most frequently reported adverse events with at least a possible relationship to saquinavir in combination with low dose ritonavir (i.e. adverse reactions) were nausea, diarrhea, fatigue, vomiting, flatulence, and abdominal pain.

mucosal ulceration, fever, wasting syndrome, allergic reaction, chest pain, shivering, edema, intoxication, parasites external, retrosternal pain and drug fever; abdominal pain.

hypertension, cyanosis, heart murmur, heart valve disorder, hypotension, syncope.

visual disturbances, taste alteration, xerophthalmia, blepharitis, earache, ear pressure, eye irritation, hearing decreased, otitis, tinnitus, dry eye syndrome.

pharyngitis, dyspnea, laryngitis, rhinitis, bronchitis, cough, epistaxis, hemoptysis, pneumonia, pulmonary disease, respiratory disorder, sinusitis, upper respiratory tract infection.

hypoaesthesia, coordination abnormal, intracranial hemorrhage, ataxia, confusion, dry mouth, convulsions, dysesthesia, tremor, dysarthria, heart rate disorder, hyperesthesia, hyperreflexia, hyporeflexia, light-headed feeling, myelopolyradiculoneuritis, numbness face, pain facial, paresis, poliomyelitis, prickly sensation, progressive multifocal leukoencephalopathy, spasms, unconsciousness, and extremity numbness.

micturition disorder, renal calculus, urinary tract bleeding, urinary tract infection.

Stevens-Johnson syndrome, dermatitis bullous, drug eruption, severe cutaneous reaction associated with increased liver function tests, sweating increased, hot flushes, skin pigment changes, acne, dermatitis, folliculitis, alopecia, and polyarthritis, chalazion, dermatitis seborrheic, eczema, erythema, furunculosis, hair changes, nail disorder, night sweats, photosensitivity reaction, rash maculopapular, skin disorder, skin nodule, skin ulceration, uticaria, verruca.

staphylococcal infection, abscess, angina tonsillaris, candidiasis, cellulitis, herpes simplex, herpes zoster, infection bacterial, infection mycotic, influenza, moniliasis.

impotence, prostate enlarged, vaginal discharge.

jaundice, portal hypertension, and exacerbation of chronic liver disease with Grade 4 elevated liver function test, hepatomegaly, hepatosplenomegaly, hyperbilirubinemia, liver enzyme disorder.

vasoconstriction and vein distended.

hemolytic anemia, neutropenia, thrombocytopenia, bleeding dermal, leucopenia, microhemorrhages, pancytopenia, splenomegaly, lymphadenopathy.

blood glucose increased, and blood glucose decreased, increased alkaline phosphatase, increased creatinine phosphokinase, increased gamma GT, raised amylase, raised LDH, TSH increase, hyperglycemia, hypercalcemia, hyperkalemia, hypernatremia, hyperphosphatemia, hypocalcemia, hypokalemia, hyponatremia, hypophosphatemia; hyperbilirubinemia.

ascites, intestinal obstruction, constipation, eructation, stomatitis, discoloured feces, glossitis, frequent bowel movements, gastralgia, gastritis, gastrointestinal inflammation, pancreatitis, pancreatitis leading to death, tooth disorder, cheilitis, colic abdominal, dysphagia, esophagitis, feces bloodstained, gingivitis, hemorrhage rectum, hemorrhoids, infectious diarrhea, melena, pain pelvic, painful defecation, parotid disorder, salivary glands disorder, stomach upset, toothache.

confusional state, suicide attempt, insomnia, euphoria, anxiety, reduced intellectual ability, irritability, agitation, hallucination, somnolence, depression, amnesia, anxiety attack, dreaming excessive, lethargy, libido disorder, overdose effect, psychic disorder, psychosis, speech disorder.

Only limited experience is available in elderly patients. No data are available to establish a dose recommendation in elderly patients.

The safety and efficacy of saquinavir in HIV-infected children has not been established.

Each hard gelatin, light brown and green capsule, imprinted with “Roche” and “0245” on opaque shells, contains: saquinavir 200 mg, present as saquinavir mesylate. Nonmedicinal ingredients: lactose, magnesium stearate, microcrystalline cellulose, povidone, sodium starch glycolate and talc; capsule shell: gelatin, indigotine, iron oxide and titanium dioxide. Glass or plastic (HDPE) bottles of 270.

Each light orange to greyish- or brownish-orange, oval cylindrical, biconvex film-coated tablet with “ROCHE” and “SQV 500” imprinted on the tablet face, contains saquinavir 500 mg, present as saquinavir mesylate. Nonmedicinal ingredients: croscarmellose sodium, lactose, magnesium stearate, microcrystalline cellulose and povidone K30; film-coat: hypromellose, iron oxide red, iron oxide yellow, talc, titanium dioxide and triacetin. Plastic (HDPE) bottles of 120.

No human data is available.

Varying degrees of cross-resistance among protease inhibitors have been observed. Continued administration of INVIRASE therapy following loss of viral suppression may increase the likelihood of cross-resistance to other protease inhibitors (see also Action and Clinical Pharmacology, Resistance).

Renal clearance is only a minor elimination pathway, the principal route of metabolism and excretion for saquinavir is via the liver. Therefore, no initial dose adjustment is necessary for patients with renal impairment. However, patients with severe renal impairment have not been studied and caution should be exercised when prescribing saquinavir in this population.

To monitor maternal-fetal outcomes of pregnant women exposed to INVIRASE and other antiretroviral drugs, an Antiretroviral Pregnancy Registry has been established. Physicians are encouraged to register patients by calling 1-800-258-4263.

Reproduction studies with saquinavir in rats have shown no embryotoxicity or teratogenicity at plasma exposures (AUC values) up to 5 times those achieved with human use (1800 mg/day), or in rabbits at dose levels up to 24 times the recommended human dose. There are however, no adequate or well controlled studies of INVIRASE in pregnant women. Because animal reproduction studies are not always predictive of human response, INVIRASE should be used during pregnancy only if the potential benefits are considered to outweigh the potential risks to the fetus.

The safety and efficacy of saquinavir in HIV-infected children has not been established. The safety and efficacy of saquinavir when coadministered with ritonavir to pediatric patients is under investigation.

Immune reconstitution syndrome has been reported in patients treated with combination antiretroviral therapy, including INVIRASE. During the initial phase of combination antiretroviral treatment, patients whose immune system responds may develop an inflammatory response to indolent or residual opportunistic infections (such as M. avium infection, cytomegalovirus, P. jiroveci pneumonia (PCP), or tuberculosis), which may necessitate further evaluation and treatment.

Elevated cholesterol and/or triglyceride levels have been observed in some patients taking saquinavir in combination with ritonavir. Cholesterol and triglyceride levels should be monitored prior to initiating combination therapy with INVIRASE/ritonavir, and at periodic intervals while on such therapy. In these patients, lipid disorders should be managed as clinically appropriate.

New onset diabetes mellitus, exacerbation of pre-existing diabetes mellitus and hyperglycemia have been reported during post-marketing surveillance in HIV-infected patients receiving protease inhibitor therapy. Some patients required either initiation or dose adjustments of insulin or oral hypoglycemic agents for treatment of these events. In some cases diabetic ketoacidosis has occurred. In those patients who discontinued protease inhibitor therapy, hyperglycemia persisted in some cases. Because these events have been reported voluntarily during clinical practice, estimates of frequency cannot be made and a causal relationship between protease inhibitor therapy and these events has not been established.

Only limited experience is available in elderly patients. No data are available to establish a dose recommendation in elderly patients.

Each capsule contains lactose (anhydrous) 63.3 mg and each film-coated tablet contains lactose (monohydrate) 38.5 mg. Patients with rare hereditary problems of galactose intolerance, the Lapp lactase deficiency or glucose-galactose malabsorption (autosomal recessive disorder) should not take this medicine.

INVIRASE should only be used if it is combined with ritonavir.

When INVIRASE is prescribed in combination with other antiretroviral therapies, physicians should refer to the appropriate Product Monographs for safety and prescribing information.

If a serious or severe toxicity occurs during treatment with INVIRASE, treatment with the drug should be interrupted until the etiology of the event is identified or the toxicity resolves. At that time, resumption of treatment with full dose may be considered.

Clinical chemistry tests, viral load and CD₄ count should be performed prior to initiating therapy and at appropriate intervals thereafter. Elevated nonfasting triglyceride levels have been observed in patients in saquinavir trials. Triglyceride levels should be periodically monitored during therapy. Increases in cholesterol have also been observed and should be monitored. For comprehensive information concerning laboratory test alterations associated with the use of other antiretroviral therapies, physicians should refer to the complete product information for these drugs.

In cases of mild impairment no initial dosage adjustment is necessary at the recommended dose. The use of INVIRASE (alone or in combination with ritonavir) in patients with moderate hepatic impairment has not been studied. In the absence of such studies, caution should be exercised, as increases in saquinavir levels and/or increases in liver enzymes may occur. In patients with underlying hepatitis B or C, cirrhosis, chronic alcoholism and/or other underlying liver abnormalities there have been reports of worsening liver disease and development of portal hypertension after starting saquinavir. Associated symptoms include jaundice, ascites, edema and, in some cases esophageal varices. Several of these patients died. A causal relationship between saquinavir therapy and development of portal hypertension has not been established. Increased monitoring for signs and symptoms of liver toxicity should be considered. INVIRASE/ritonavir is contraindicated in patients with severe hepatic impairment (see Contraindications).

Elevated cholesterol and/or triglyceride levels have been observed in some patients taking saquinavir in combination with ritonavir. Marked elevation in triglyceride levels is a risk factor for development of pancreatitis (see Warnings and Precautions, Cardiac Disorders).

Redistribution/accumulation of body fat including central obesity, dorsocervical fat enlargement (buffalo hump), peripheral wasting, facial wasting, breast enlargement, and “Cushingoid appearance” have been observed in patients receiving antiretroviral therapy. The mechanism and long-term consequences of these events are currently unknown. A causal relationship has not been established.

There have been reports of increased bleeding including spontaneous skin hematomas and hemarthrosis in patients with Hemophilia Type A and Type B treated with protease inhibitors. In some patients, additional Factor VIII was given. In more than half of the reported cases, treatment with protease inhibitors was continued or re-introduced. There is no proven relationship between protease inhibitors and such bleeding, however, the frequency of bleeding episodes should be closely monitored in patients on saquinavir.

It is not known whether saquinavir is excreted in human milk. Because many drugs are excreted in human milk, it is advisable to caution mothers against breast feeding while taking INVIRASE. Animal studies indicate that administration of saquinavir to rats through the lactation period at plasma concentrations (AUC values) up to 5 times those achieved with the human-dose (1800 mg/day) had no effect on the survival, growth or development of offspring to weaning. However, the potential for adverse reactions to saquinavir in nursing infants cannot be assessed. Current medical practice advises against breast-feeding by HIV-infected women, due to the possibility of post-natal transmission.

The mean steady-state volume of distribution following intravenous administration of a 12 mg dose of saquinavir is 700 L (CV 39%), indicating extensive partitioning into tissues. Saquinavir shows a high degree of protein binding (~98%), which is independent of concentration over the range 15-700 ng/mL.

In two patients treated with saquinavir (600 mg TID), cerebrospinal fluid concentrations were negligible when compared to concentrations from matching plasma samples.

The pharmacokinetic properties of saquinavir have been evaluated in healthy volunteers and in HIV-infected patients after single and multiple oral doses of 25, 75, 200 and 600 mg TID; and in healthy volunteers after intravenous infusion doses of 12 mg administered over 1 hour, and 6, 36 and 72 mg administered over 3 hours.

In vitro selection of resistance from wild-type HIV-1 virus: The most commonly reported mutations, G48V and L90M, were observed to develop during in vitro passage of HIV-1 wild-type virus in the presence of increasing concentrations of saquinavir. Recombinant virus harboring the G48V and L90M mutations exhibited 7.9-fold and 3.3-fold reductions in viral susceptibility to saquinavir, respectively. Protease mutations such as M36I, I54V, K57R, and L63V developed less frequently in the presence of saquinavir.

Renal excretion is a very minor route of elimination for saquinavir (<4%). Systemic clearance is rapid, 80 L/h, which is close to hepatic plasma flow. Systemic clearance was constant after intravenous doses of 6, 36 and 72 mg infused over 3 hours. The mean residence time of the drug was found to be 7 hours.

In a mass balance study using ¹⁴C-saquinavir (n=8), 88% and 1% of the orally administered radioactivity was recovered in feces and urine, respectively, within 4 days of dosing. In an additional four subjects administered 10.5 mg ¹⁴C-saquinavir intravenously, 81% and 3% of the intravenously administered radioactivity was recovered in the feces and urine, respectively, within 4 days of dosing. In mass-balance studies, 13% of circulating saquinavir in plasma was present as unchanged drug after oral administration and the remainder present as metabolites.

Pharmacokinetic Parameters of Saquinavir at Steady-State After Administration of Different Regimens in HIV-Infected Patients. Arithmetic Mean ± Standard Deviation

A gender difference was observed, with females showing a higher saquinavir exposure than males (mean AUC increase of 56%, mean C_max increase of 26%), in the bioequivalence study comparing INVIRASE 500 mg film coated tablets to the INVIRASE 200 mg capsules in combination with ritonavir. There was no evidence that age and body weight explained the gender difference in this study. A clinically significant difference in safety and efficacy between men and women has not been reported with the 1000 mg INVIRASE/100 mg ritonavir b.i.d. dosage regimen.

Greater than 96% of a radiolabelled I.V. dose appears in the feces within 48 hours, indicating extensive hepatic clearance. Hepatic metabolism is P450-mediated, of which >90% is the work of one isozyme (CYP3A4). The metabolic profile of saquinavir has been investigated in bile, plasma and microsomes in rats and in microsomes from other species, including man. Saquinavir is rapidly metabolized to a range of mono- and di-hydroxylated inactive compounds. Following intravenous administration, 66% of circulating saquinavir is present as unchanged drug and the remainder as metabolites, suggesting that saquinavir undergoes extensive first-pass metabolism.

Four studies have investigated ritonavir-boosted saquinavir regimens in ART naïve patients [(saquinavir/ritonavir 1600 mg/100 mg qd (n=349); 1000 mg/100 mg bid (n=92)]. Baseline resistance analyses were conducted on 26 patients experiencing virological rebound. Data from two patients was excluded either because PI mutations were present at baseline or a signature protease mutation (D30N) associated with another PI subsequently developed. Virus from two patients (2/24) developed protease mutations (M36I and M46i/m, respectively). These mutations are not typically associated with saquinavir resistance. No specific saquinavir-associated protease mutations were observed to develop following virological failure.

Baseline and on-therapy genotype was evaluated for 22 previously PI-experienced patients who experienced virological failure after receiving a ritonavir-boosted saquinavir regimen (MaxCmin 1 and 2 studies; 1000/100 mg bid, n=171). Virus from eight patients (8/22; 36%) developed additional protease mutations following virological failure. The relative incidence of each mutation was: I84V (n=4, 18%); F53L, A71V or G73S (n=2, 9%); L10V, M46I, I54V, V82A or L90M (n=1, 4.5%).

The pharmacokinetics of saquinavir when administered as INVIRASE have not been sufficiently investigated in pediatric patients.

In cases of mild impairment no initial dosage adjustment is necessary at the recommended dose. The use of INVIRASE (alone or in combination with ritonavir) in patients with moderate hepatic impairment has not been studied. In the absence of such studies, caution should be exercised, as increases in saquinavir levels and/or increases in liver enzymes may occur. In patients with underlying hepatitis B or C, cirrhosis, chronic alcoholism and/or other underlying liver abnormalities there have been reports of worsening liver disease and development of portal hypertension after starting saquinavir. Associated symptoms include jaundice, ascites, edema and, in some cases esophageal varices. Several of these patients died. A causal relationship between saquinavir therapy and development of portal hypertension has not been established. Increased monitoring for signs and symptoms of liver toxicity should be considered (see Contraindications).

The pharmacokinetics of saquinavir when administered as INVIRASE have not been sufficiently investigated in patients >65 years of age.

Saquinavir is a selective and reversible inhibitor of HIV protease, an essential viral enzyme which is required for specific cleavage of viral gag and gag-pol polyproteins and ultimately, for the release of mature, infectious virus.

Antiviral activity in vitro: Saquinavir demonstrates antiviral activity against a panel of laboratory strains and clinical isolates of HIV-1 with typical EC₅₀ and EC₉₀ values in the range 1-10 nM and 5-50 nM. In the presence of 50% human serum or alpha-1 glycoprotein (1 mg/mL), the antiviral activity of saquinavir decreases by an average factor of 25-fold and 14-fold, respectively. There is no apparent difference in the antiviral activity of saquinavir between subtype B and non-B clades whereas EC₅₀ values are in the range of 0.3-2.4 nM with clinical isolates of HIV-2.

The influence of race on the pharmacokinetics of INVIRASE has not been determined.

Renal clearance is only a minor elimination pathway, the principal route of metabolism and excretion for saquinavir is via the liver. Therefore, no initial dose adjustment is necessary for patients with renal impairment. However, patients with severe renal impairment have not been studied and caution should be exercised when prescribing saquinavir in this population.